Interferon tau compositions and methods of use

ABSTRACT

The present invention describes the production of interferon-τ proteins and polypeptides derived therefrom. The antiviral and anticellular proliferation properties of these proteins and polypeptides are disclosed. One advantage of the proteins of the present invention is that they do not have cytotoxic side-effects when used to treat cells. Structure/function relationships for the interferon-τ protein are also described. In one aspect, the invention includes ovine interferon-τ. In another aspect the invention includes multiple forms of human interferon-τ.

[0001] This application is a continuation of patent application Ser. No.08/455,021, filed May 31, 1995, which is a continuation of applicationSer. No. 08/438,753, filed May 10, 1995, now U.S. Pat. No. 5,705,363,which is a continuation-in-part of application Ser. No. 08/139,891,filed Oct. 19, 1993, now abandoned. patent application Ser. No.08/139,891 was incorporated by reference into application Ser. No.08/438,753 (now U.S. Pat. No. 5,705,363), which was incorporated byreference into application Ser. No. 08/455,021, which is incorporated byreference herein. Portions of the Ser. No. 08/139,891 application whichwere incorporated by reference into the intervening applications, andthus into the present application, are specifically included herein.

[0002] This invention was made with government support under NationalInstitutes of Health grants HD 10436, HD 26006, CA 38587, and CA 57084.Accordingly, the United States government has certain rights in thisinvention.

FIELD OF THE INVENTION

[0003] The present invention relates to interferon-τ compositions andmethods of use.

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BACKGROUND OF THE INVENTION

[0115] Conceptus membranes, or trophectoderm, of various mammals producebiochemical signals that allow for the establishment and maintenance ofpregnancy (Bazer, et al., 1983). One such protein, ovine trophoblastprotein-one (oTP-1), was identified as a low molecular weight proteinsecreted by sheep conceptuses between days 10 and 21 of pregnancy(Wilson, et al., 1979; Bazer, et al., 1986). The protein oTP-1 was shownto inhibit uterine secretion of prostaglandin F₂-alpha, which causes thecorpus luteum on the ovary to undergo physiological and endocrinologicaldemise in nonpregnant sheep (Bazer, et al., 1986). Accordingly, oTP-1has antiluteolytic biological activity. The primary role of oTP-1 wasassumed to be associated with the establishment of pregnancy.

[0116] oTP-1 was subsequently found to (i) exhibit limited homology(50-70%) with interferon alphas (IFNα) of various species (Imakawa, etal., 1987), and (ii) bind to a Type I interferon receptor (Stewart, etal., 1987). Despite some similarities with IFNα, oTP-1 has severalfeatures that distinguish it from IFNα including the following: oTP-1'srole in reproductive biochemistry (other interferons are not known tohave any role in the biochemical regulation of reproductive cycles),oTP-1's cellular source—trophoblast cells (IFNα is derived fromlymphocyte cells), oTP-1's size—172 amino acids (IFNα is typically about166 amino acids), and oTP-1 is weakly inducible by viruses (IFNα ishighly inducible by viruses). The International Interferon Societyrecognizes oTP-1 as belonging to an entirely new class of interferonswhich have been named interferon-tau (IFNτ). The Greek letter τ standsfor trophoblast.

[0117] The interferons have been classified into two distinct groups:type I interferons, including IFNα, IFNβ, and IFNω (also known asIFNαII); and type II interferons, represented by IFNγ (reviewed byDeMaeyer, et al., 1988). In humans, it is estimated that there are atleast 17 IFNα non-allelic genes, at least about 2 or 3 IFNβ non-allelicgenes, and a single IFNγ gene.

[0118] IFNα's have been shown to inhibit various types of cellularproliferation. IFNα's are especially useful against hematologicmalignancies such as hairy-cell leukemia (Quesada, et al., 1984).Further, these proteins have also shown activity against multiplemyeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi'ssarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinarybladder tumors and ovarian cancers (Bonnem, et al., 1984; Oldham, 1985).The role of interferons and interferon receptors in the pathogenesis ofcertain autoimmune and inflammatory diseases has also been investigated(Benoit, et al., 1993).

[0119] IFNα's are also useful against various types of viral infections(Finter, et al., 1991). Alpha interferons have shown activity againsthuman papillomavirus infection, Hepatitis B, and Hepatitis C infections(Finter, et al., 1991; Kashima, et al., 1988; Dusheiko, et al., 1986;Davis, et al., 1989).

[0120] Significantly, however, the usefulness of IFNα's has been limitedby their toxicity: use of interferons in the treatment of cancer andviral disease has resulted in serious side effects, such as fever,chills, anorexia, weight loss, and fatigue (Pontzer, et al., 1991;Oldham, 1985). These side effects often require (i) the interferondosage to be reduced to levels that limit the effectiveness oftreatment, or (ii) the removal of the patient from treatment. Suchtoxicity has reduced the usefulness of these potent antiviral andantiproliferative proteins in the treatment of debilitating human andanimal diseases.

SUMMARY OF THE INVENTION

[0121] In a first aspect, the present invention relates to compositionsof and methods employing ovine interferon-τ. The invention includes anisolated nucleic acid molecule that encodes an ovine interferon-τ. Oneembodiment of this nucleic acid molecule is a nucleic acid moleculehaving the sequence presented as SEQ ID NO:1. In another embodiment, thenucleic acid molecule encodes an ovine interferon-τ polypeptide having asequence presented as SEQ ID NO:2. The ovine interferon-τ polypeptidemay include an amino-terminal extension, such as, a leader sequence.

[0122] In another embodiment, the present invention includes anexpression vector having a nucleic acid containing an open reading frame(ORF) that encodes an ovine interferon-τ, including the nucleic acid andpolypeptide sequences described above. The vector further includesregulatory sequences effective to express the open reading frame in ahost cell. Further, the invention includes a method of recombinantlyproducing ovine interferon-τ using the expression vectors of the presentinvention. The expression vectors are introduced into suitable hostcells. The host cells are then cultured under conditions that result inthe expression of the ORF sequence.

[0123] In one embodiment, the present invention includes a recombinantlyproduced ovine interferon-τ protein.

[0124] Further, the invention includes a method of inhibiting tumor cellgrowth. In the method, the tumor cells are contacted with ovineinterferon-τ at a concentration effective to inhibit growth of the tumorcells. Target tumor cells include, but are not limited to carcinomacells, hematopoietic cancer cells, leukemia cells, lymphoma cells andmelanoma cells.

[0125] The invention also includes a method of inhibiting viralreplication. In this method, cells infected with a virus are contactedwith ovine interferon-τ at a concentration effective to inhibit viralreplication within said cells. Ovine interferon-τ may be used to inhibitthe replication of both RNA and DNA viruses. Exemplary RNA virusesinclude feline leukemia virus, ovine progressive pneumonia virus, ovinelentivirus, equine infectious anemia virus, bovine immunodeficiencyvirus, visna-maedi virus, and caprine arthritis encephalitis virus.

[0126] In a second aspect, the present invention relates to compositionsof and methods employing human interferon-τ's. In one embodiment, theinvention includes an isolated nucleic acid molecule that encodes ahuman interferon-τ. Several variants of human interferon-τ (HuIFNτ) aredisclosed herein, including HuIFNτ1, HuIFNτ2, HuIFNτ3, HuIFNτ4, HuIFNτ5,HuIFNτ6 and HuIFNτ7.

[0127] The nucleic acid molecules of the present invention includenucleic acid molecules having the following sequences: SEQ ID NO:43, SEQID NO:29, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:27, SEQ ID NO:21 and SEQID NO:23.

[0128] The nucleic acids of the present invention also include nucleicacid molecules encoding the following polypeptide sequences: SEQ IDNO:44, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:22, and SEQ ID NO:24. The nucleic acids may further include sequencesencoding leader sequences for the human interferon-τ which they encode,for example, SEQ ID NO:41 or SEQ ID NO:42.

[0129] The second aspect of the invention further includes an expressionvector having a nucleic acid sequence containing an open reading framethat encodes a human interferon-τ, including the nucleic acid andpolypeptide sequences described above. The vector further includesregulatory sequences effective to express said open reading frame in ahost cell. The regulatory sequence may include sequences useful fortargeting or secretion of the human IFNτ polypeptide: such sequences maybe endogenous (such as the normally occurring human IFNτ leadersequences, present, for example, in SEQ ID NO:41) or heterologous (suchas a secretory signal recognized in yeast, mammalian cell, insect cell,tissue culture or bacterial expression systems). In the expressionvector, regulatory sequences may also include, 5′ to said nucleic acidsequence, a promoter region and an ATG start codon in-frame with thehuman interferon-τ coding sequence, and 3′ to said coding sequence, atranslation termination signal followed by a transcription terminationsignal.

[0130] In a further embodiment, the invention includes a method ofrecombinantly producing human interferon-τ. In the method, theexpression vector, containing sequences encoding a human interferon-τopen reading frame (ORF), is introduced into suitable host cells, wherethe vector is designed to express the ORF in the host cells. Thetransformed host cells are then cultured under conditions that result inthe expression of the ORF sequence. Numerous vectors and theircorresponding hosts are useful in the practice of this method of theinvention, including, lambda gt11 phage vector and E. coli cells. Otherhost cells include, yeast, mammalian cell, insect cell, tissue culture,plant cell culture, transgenic plants or bacterial expression systems.

[0131] In another embodiment, the invention includes an isolated humaninterferon-τ protein or polypeptide. The protein may be recombinantlyproduced. Further, the protein or polypeptide may include any of thefollowing human interferon-τ sequences: SEQ ID NO:44, SEQ ID NO:30, SEQID NO:34, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:22, and SEQ ID NO:24.

[0132] The invention further includes a method of inhibiting tumor cellgrowth. In the method, the tumor cells are contacted with a humaninterferon-τ polypeptide at a concentration effective to inhibit growthof the tumor cells. The human interferon-τ may be a part of anyacceptable pharmacological formulation. Tumor cells whose growth may beinhibited by human interferon-τ include, but are not limited to, humancarcinoma cells, hematopoietic cancer cells, human leukemia cells, humanlymphoma cells, and human melanoma cells. In one embodiment, the tumorcells are steroid-sensitive tumor cells, for example, mammary tumorcells.

[0133] In yet another embodiment of the present invention, humaninterferon-τ polypeptides are used in a method of inhibiting viralreplication. In this method, cells infected with a virus are contactedwith human interferon-τ at a concentration effective to inhibit viralreplication within said cells. The human interferon-τ may be a part ofany acceptable pharmacological formulation. The replication of both RNAand DNA viruses may be inhibited by human interferon-τ polypeptides.Exemplary RNA viruses include human immunodeficiency virus (HIV) orhepatitis c virus (HCV). An exemplary DNA virus is hepatitis B virus(HBV).

[0134] In yet another aspect, the present invention includes a method ofenhancing fertility in a female mammal. In this method, an effectivemammalian fertility enhancing amount of human interferon-τ isadministered to the female mammal in a pharmaceutically acceptablecarrier.

[0135] The invention also includes isolated human interferon-τpolypeptides. These polypeptides are derived from the interferon-τ aminoacid sequence and are typically between about 15 and 172 amino acids inlength.

[0136] The invention also includes hybrid α-interferon molecules inwhich the toxicity portion of native IFNα has been replaced by analogoussequences from IFNτ.

[0137] Also included in the invention is a fusion polypeptide thatcontains a human interferon-τ polypeptide that is between 15 and 172amino acids long and derived from a human interferon-τ amino acid codingsequence, and a second soluble polypeptide. In one embodiment, humaninterferon-τ sequences are used in fusion constructs with other types ofinterferons to reduce the toxicity of the other types of interferons,for example, interferon-α and interferon-β.

[0138] The invention also includes a polypeptide composition having (a)a human interferon-τ polypeptide, where said polypeptide is (i) derivedfrom an interferon-τ amino acid coding sequence, and (ii) between 15 and172 amino acids long, and (b) a second soluble polypeptide. Interferon-αand interferon-β are examples of such second soluble polypeptides. Thiscomposition may be used to reduce the toxicity of the other types ofinterferons when the interferons are used in pharmaceutical formulationsor in therapeutic applications.

[0139] The invention also includes purified antibodies that areimmunoreactive with human interferon-τ. The antibodies may be polyclonalor monoclonal.

[0140] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0141]FIGS. 1A and 1B present the nucleic acid coding sequence of asynthetic gene of OvIFNτ designed to include 19 unique restrictionenzyme sites spaced evenly throughout the coding sequence.

[0142]FIG. 2 shows the cloning strategy used for making a synthetic geneencoding OvIFNτ.

[0143]FIG. 3 shows a comparison of the predicted protein sequences of ahuman interferon-τ gene and an ovine interferon-τ gene. Divergent aminoacids are indicated by presentation of the alternative amino acid on theline below the nucleic acid sequences.

[0144]FIG. 4 presents data demonstrating that both OvIFNτ and IFNα wereable to drastically reduce growth of HL-60 cells.

[0145]FIG. 5 presents data demonstrating that rHuIFNα is cytotoxic andOvIFNτ is not. In the figure, results of one of three replicateexperiments are presented as mean % viability±SD.

[0146]FIG. 6 presents the sequences of polypeptides derived from theIFNτ sequence.

[0147]FIG. 7 presents the complete nucleic acid and amino acid sequenceof an OvIFNτ sequence.

[0148]FIG. 8 presents data supporting the lack of cytotoxicity, relativeto IFNα, when IFNτ is used to treat peripheral blood mononuclear cells.

[0149]FIG. 9 shows the results of treatment of a human cutaneous T celllymphoma line, HUT 78, with IFNτ.

[0150]FIG. 10 shows the results of treatment of a human T cell lymphomaline, H9, with IFNτ.

[0151]FIG. 11A presents data for the peptide inhibition, relative to FIV(feline immunodeficiency virus) replication, of polypeptides derivedfrom OvIFNτ with whole OvIFNτ. FIG. 11B presents data for the peptideinhibition, relative to HIV (human immunodeficiency virus) replication,of polypeptides derived from OvIFNτ with whole OvIFNτ.

[0152]FIG. 12 presents data demonstrating the inhibition of theantiviral activity of IFNτ by IFNτ-derived peptides.

[0153]FIG. 13 presents data demonstrating the inhibition by IFNτ-derivedpeptides of OvIFNτ antiviral activity.

[0154]FIG. 14 presents data demonstrating the inhibition by IFNτ-derivedpeptides of bovine IFNα antiviral activity.

[0155]FIG. 15 presents data demonstrating the inhibition by IFNτ-derivedpeptides of human IFNα antiviral activity.

[0156]FIG. 16 presents data evaluating the lack of inhibition byIFNτ-derived peptides of bovine IFNγ antiviral activity.

[0157]FIG. 17 presents data demonstrating the anti-IFNτ-derived peptideantisera inhibition of the antiviral activity of IFNτ.

[0158]FIG. 18 presents data demonstrating the anti-IFNτ-derived peptideantisera inhibition of the binding of radiolabeled IFNτ to cells.

[0159]FIGS. 19A and 19B present an alignment of nucleic acid sequencesencoding IFNτ polypeptides.

[0160]FIGS. 20A and 20B present an alignment of amino acid sequences ofIFNτ polypeptides.

[0161]FIG. 21 presents data comparing the cytotoxicity of IFNτ withIFNβ.

BRIEF DESCRIPTION OF THE SEQUENCES

[0162] SEQ ID NO:1 is the nucleotide sequence of a synthetic geneencoding ovine interferon-τ (OvIFNτ). Also shown is the encoded aminoacid sequence.

[0163] SEQ ID NO:2 is an amino acid sequence of a mature OvIFNτ protein.

[0164] SEQ ID NO:3 is a synthetic nucleotide sequence encoding a maturehuman interferon-τ (HuIFNτ) protein.

[0165] SEQ ID NO:4 is an amino acid sequence for a mature HuIFNτ1protein.

[0166] SEQ ID NO:5 is the amino acid sequence of fragment 1-37 of SEQ IDNO:2.

[0167] SEQ ID NO:6 is the amino acid sequence of fragment 34-64 of SEQID NO:2.

[0168] SEQ ID NO:7 is the amino acid sequence of fragment 62-92 of SEQID NO:2.

[0169] SEQ ID NO:8 is the amino acid sequence of fragment 90-122 of SEQID NO:2.

[0170] SEQ ID NO:9 is the amino acid sequence of fragment 119-150 of SEQID NO:2.

[0171] SEQ ID NO:10 is the amino acid sequence of fragment 139-172 ofSEQ ID NO:2.

[0172] SEQ ID NO:11 is the nucleotide sequence of a natural HuIFNτ1 genewith a leader sequence.

[0173] SEQ ID NO:12 is the predicted amino acid coding sequence of theSEQ ID NO:11.

[0174] SEQ ID NO:13 is a 25-mer synthetic oligonucleotide according tothe subject invention.

[0175] SEQ ID NO:14 is a 25-mer synthetic oligonucleotide according thesubject invention.

[0176] SEQ ID NO:15 is the amino acid sequence of fragment 1-37 of SEQID NO:4.

[0177] SEQ ID NO:16 is the amino acid sequence of fragment 34-64 of SEQID NO:4.

[0178] SEQ ID NO:17 is the amino acid sequence of fragment 62-92 of SEQID NO:4.

[0179] SEQ ID NO:18 is the amino acid sequence of fragment 90-122 of SEQID NO:4.

[0180] SEQ ID NO:19 is the amino acid sequence of fragment 119-150 ofSEQ ID NO:4.

[0181] SEQ ID NO:20 is the amino acid sequence of fragment 139-172 ofSEQ ID NO:4.

[0182] SEQ ID NO:21 is the nucleotide sequence of cDNA HuIFNτ6.

[0183] SEQ ID NO:22 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:21.

[0184] SEQ ID NO:23 is the nucleotide sequence of cDNA HuIFNτ7.

[0185] SEQ ID NO:24 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:23.

[0186] SEQ ID NO:25 is the nucleotide sequence of cDNA HuIFNτ4.

[0187] SEQ ID NO:26 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:25.

[0188] SEQ ID NO:27 is the nucleotide sequence of cDNA HuIFNτ5.

[0189] SEQ ID NO:28 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:27.

[0190] SEQ ID NO:29 is the nucleotide sequence of genomic DNA cloneHuIFNτ2.

[0191] SEQ ID NO:30 is the predicted amino acid sequence encoded by thesequence represented as SEQ ID NO:29.

[0192] SEQ ID NO:31 is the nucleotide sequence, including leadersequence, of genomic DNA clone HuIFNτ3, a natural HuIFNτ gene.

[0193] SEQ ID NO:32 is the predicted amino acid sequence (includingleader sequence) encoded by the sequence represented as SEQ ID NO:31.

[0194] SEQ ID NO:33 is the nucleotide sequence, excluding leadersequence, of genomic DNA clone HuIFNτ3, a natural HuIFNτ gene.

[0195] SEQ ID NO:34 is the predicted amino acid sequence of a maturehuman IFNτ protein encoded by HuIFNτ3, encoded by the sequencerepresented as SEQ ID NO:33.

[0196] SEQ ID NO:35 is the amino acid sequence of fragment 1-37 of SEQID NO:33.

[0197] SEQ ID NO:36 is the amino acid sequence of fragment 34-64 of SEQID NO:33.

[0198] SEQ ID NO:37 is the amino acid sequence of fragment 62-92 of SEQID NO:33.

[0199] SEQ ID NO:38 is the amino acid sequence of fragment 90-122 of SEQID NO:33.

[0200] SEQ ID NO:39 is the amino acid sequence of fragment 119-150 ofSEQ ID NO:33.

[0201] SEQ ID NO:40 is the amino acid sequence of fragment 139-172 ofSEQ ID NO:33.

[0202] SEQ ID NO:41 is the amino acid sequence of fragment 1-23 of SEQID NO:32.

[0203] SEQ ID NO:42 is the amino acid sequence of fragment 1-23 of SEQID NO:11.

[0204] SEQ ID NO:43 is the nucleotide sequence, excluding leadersequence, of DNA clone HuIFNτ1.

[0205] SEQ ID NO:44 is the predicted amino acid sequence of a maturehuman IFNτ protein encoded by HuIFNτ1, encoded by the sequencerepresented as SEQ ID NO:43.

DETAILED DESCRIPTION OF THE INVENTION

[0206] I. Definitions

[0207] Interferon-τ (IFNτ) refers to any one of a family of interferonproteins having greater than 70%, or preferably greater than about 80%,or more preferably greater than about 90% amino acid homology to eitherthe sequence presented as (a) SEQ ID NO:2 or (b) SEQ ID NO:34. Aminoacid homology can be determined using, for example, the LALIGN programwith default parameters. This program is found in the FASTA version 1.7suite of sequence comparison programs (Pearson, et al., 1988; Pearson,1990; program available from William R. Pearson, Department ofBiological Chemistry, Box 440, Jordan Hall, Charlottesville, Va.).

[0208] Typically, IFNτ has at least one characteristic from thefollowing group of characteristics: (a) expressed during embryonic/fetalstages by trophectoderm/placenta, (b) anti-luteolytic properties, (c)anti-viral properties, and (d) anti-cellular proliferation properties.IFNτ can be obtained from a number of sources including cows, sheep, ox,and humans.

[0209] An interferon-τ polypeptide is a polypeptide having between about15 and 172 amino acids derived from an interferon-τ amino acid codingsequence, where said 15 to 172 amino acids are contiguous in nativeinterferon-τ. Such 15-172 amino acid regions can also be assembled intopolypeptides where two or more such interferon-τ regions are joined thatare normally discontinuous in the native protein.

[0210] II. Isolation & Characterization of Interferon-τ.

[0211] A. Ovine and Bovine Interferon-τ.

[0212] 1. Interferon-τ Coding Sequences.

[0213] Ovine interferon-τ (OvIFNτ) is a major conceptus secretoryprotein produced by the embryonic trophectoderm during the criticalperiod of maternal recognition in sheep. One isolate of mature OvIFNτ is172 amino acids in length (SEQ ID NO:2). The cDNA coding sequencecontains an additional 23 amino acids at the amino-terminal end of themature protein (Imakawa, et al., 1987). The coding sequence of thisOvIFNτ isolate is presented as FIG. 7.

[0214] Relative to other interferons, oIFNτ shares about 45 to 68% aminoacid homology with Interferon-α and the greatest sequence similaritywith the interferon-ωs (IFNωs) of about 68%.

[0215] For the isolation of OvIFNγ protein, conceptuses were collectedfrom pregnant sheep and cultured in vitro in a modified MinimumEssential Medium as described previously (Godkin, et al., 1982).Conceptuses were collected on various days of pregnancy with the firstday of mating being described as Day 0. OvIFNτ was purified fromconceptus culture medium essentially as described by Vallet, et al.,(1987) and Godkin, et al. (1982).

[0216] The homogeneity of OvIFNτ was assessed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE; Maniatis, et al.; Ausubel,et al.). Determination of protein concentration in purified OvIFNτsamples was performed using the bicinchoninic (BCA) assay (PierceChemical Co., Rockford, Ill.; Smith, et al., 1985).

[0217] A homologous protein to OvIFNτ was isolated from cows (BoIFNτ;Helmer, et al., 1987; Imakawa, et al., 1989). OvIFNτ and BoIFNτ (i) havesimilar functions in maternal recognition of pregnancy, and (ii) share ahigh degree of amino acid and nucleotide sequence homology betweenmature proteins. The nucleic acid sequence homology between OvIFNτ andBoIFNτ is 76.3% for the 5′ non-coding region, 89.7% for the codingregion, and 91.9% for the 3′ non-coding region. The amino acid sequencehomology is 80.4%.

[0218] Example 1 describes the reproductive functions of OvIFNτ. OvIFNτand recombinant human Interferon-α2 (rHuIFNα) were infused into uterinelumen of ewes at a variety of concentrations. The life span of thecorpus luteum was assessed by examination of interestrous intervals,maintenance of progesterone secretion, and inhibition of prostaglandinsecretion (Davis, et al., 1992). Comparison of the data resulting fromthese examinations demonstrated a considerable lengthening of theinterestrous interval when OvIFNτ is administered at 100 μg/day and nomeaningful effect when rHuIFNα is administered. These data support theconclusion that OvIFNτ significantly influences the biochemical eventsof the estrous cycle.

[0219] The antiviral properties of interferon-τ at various stages of thereproductive cycle were also examined (Example 2). Conceptus cultureswere established using conceptus obtained from sheep at days 12 through16 of the estrus cycle. Antiviral activity of supernatant from eachconceptus culture was assessed. Culture supernatants had increasingantiviral activity associated with advancing development of theconceptus up to Day 16 post estrus.

[0220] 2. Recombinant Production of IFNτ

[0221] Recombinant OvIFNτ was produced using bacterial and yeast cells.The amino acid coding sequence for OvIFNτ was used to generate acorresponding DNA coding sequence with codon usage optimized forexpression in E. coli (Example 3). The DNA coding sequence wassynthetically constructed by sequential addition of oligonucleotides.Cloned oligonucleotides were fused into a single polynucleotide usingthe restriction digestions and ligations outlined in FIG. 2. Thepolynucleotide coding sequence had the sequence presented as SEQ IDNO:1.

[0222] For expression of recombinant OvIFNτ, this synthetic codingsequence can be placed in a number of bacterial expression vectors: forexample, lambda gt11 (Promega, Madison Wis.); pGEX (Smith, et al.);pGEMEX (Promega); and pBS (Stratagene, La Jolla Calif.) vectors. Otherbacterial expression vectors containing suitable promoters, such as theT7 RNA polymerase promoter or the tac promoter, may also be used.Cloning of the OvIFNτ synthetic polynucleotide into a modified pIN IIIomp-A expression vector is described in Example 3. Production of theOvIFNτ protein was induced by the addition of IPTG. Soluble recombinantIFNτ was liberated from the cells by sonication or osmoticfractionation.

[0223] The protein can be further purified by standard methods,including size fractionation (column chromatography or preoperative gelelectrophoresis) or affinity chromatography (using, for example,anti-OvIFNτ antibodies (solid support available from Pharmacia,Piscataway N.J.). Protein preparations can also be concentrated by, forexample, filtration (Amicon, Danvers, Mass.).

[0224] The synthetic OvIFNτ gene was also cloned into the yeast cloningvector pBS24Ub (Example 4; Sabin, et al.; Ecker, et al.). Syntheticlinkers were constructed to permit in-frame fusion of the OvIFNτ codingsequences with the ubiquitin coding sequences in the vector. Theresulting junction allowed in vivo cleavage of the ubiquitin sequencesfrom the OvIFNτ sequences.

[0225] The recombinant plasmid pBS24Ub-IFNτ was transformed into theyeast S. cerevisiae. Transformed yeast cells were cultured, lysed andthe recombinant IFNτ (r-IFNτ) protein isolated from the cell lysates.

[0226] The amount of r-IFNτ was quantified by radioimmunoassay.Microsequencing of the purified r-IFNτ was carried out. The resultsdemonstrated identity with native OvIFNτ through the first 15 aminoacids. The results also confirmed that the ubiquitin/IFNτ fusion proteinwas correctly processed in vivo.

[0227] Recombinant IFNτ obtained by this method exhibited antiviralactivity similar to the antiviral activity of IFNτ purified fromconceptus-conditioned culture medium.

[0228] Other yeast vectors can be used in the practice of the presentinvention. They include 2 micron plasmid vectors (Ludwig, et al.), yeastintegrating plasmids (YIps; e.g., Shaw, et al.), YEP vectors (Shen, etal.), yeast centromere plasmids (YCps; e.g., Ernst), and the like.Preferably, the vectors include an expression cassette containing aneffective yeast promoter, such as the MFα1 promoter (Ernst, Bayne, etal.), GADPH promoter (glyceraldehyde-3-phosphate-dehydrogenase; Wu, etal.), the galactose-inducible GAL10 promoter (Ludwig, et al., Feher, etal., Shen, et al.), or the methanol-regulated alcohol oxidase (AOX)promoter (Tschopp, et al.). The AOX promoter is particularly useful inPichia pastoris host cells (for example, the AOX promoter is used inpHIL and pPIC vectors included in the Pichia expression kit, availablefrom Invitrogen, San Diego, Calif.).

[0229] The expression cassette may include additional elements tofacilitate expression and purification of the recombinant protein,and/or to facilitate the insertion of the cassette into a vector or ayeast chromosome. For example, the cassette may include a signalsequence to direct secretion of the protein. An exemplary signalsequence suitable for use in a variety of yeast expression vectors isthe MFα1 pre-pro signal sequence (Bayne, et al., Ludwig, et al., Shaw,et al.). Other signal sequences may also be used. For example, the Pholsignal sequence (Elliot, et al.) is particularly effective in PichiaPastoris and Schizosaccharomyces pombe host cells.

[0230] Exemplary expression cassettes include (i) a cassette containing(5′ to 3′) the AOX promoter, the Phol signal sequence, and a DNAsequence encoding OvIFNτ, for expression in P. pastoris host cells, and(ii) a cassette containing (5′ to 3′) the MFα1 promoter, the MFα1pre-pro signal sequence, and a DNA sequence encoding IFNτ, forexpression in S. cerevisiae host cells.

[0231] Additional yeast vectors suitable for use with the presentinvention include, but are not limited to, other vectors withregulatable expression (Hitzeman, et al.; Rutter, et al.; Oeda, et al.).The yeast transformation host is typically Saccharomyces cerevisiae,however, as illustrated above, other yeast suitable for transformationcan be used as well (e.g., Schizosaccharomiyces pombe, Pichia pastorisand the like).

[0232] The DNA encoding the IFNτ polypeptide can be cloned into anynumber of commercially available vectors to generate expression of thepolypeptide in the appropriate host system. These systems include theabove described bacterial and yeast expression systems as well as thefollowing: baculovirus expression (Reilly, et al.; Beames, et al.;Clontech, Palo Alto Calif.); plant cell expression, transgenic plantexpression (e.g., S. B. Gelvin and R. A. Schilperoot, Plant MolecularBiology, 1988), and expression in mammalian cells (Clontech, Palo AltoCalif.; Gibco-BRL, Gaithersburg Md.). These recombinant polypeptides canbe expressed as fusion proteins or as native proteins. A number offeatures can be engineered into the expression vectors, such as leadersequences which promote the secretion of the expressed sequences intoculture medium. The recombinantly produced polypeptides are typicallyisolated from lysed cells or culture media. Purification can be carriedout by methods known in the art including salt fractionation, ionexchange chromatography, and affinity chromatography. Immunoaffinitychromatography can be employed, as described above, using antibodiesgenerated based on the IFNτ polypeptides.

[0233] B. Human Interferon-τ

[0234] 1. Identification and Cloning of Human Genomic Sequences Encodingan Interferon-τ Protein.

[0235] Human genomic DNA was screened for sequences homologous tointerferon-τ (Example 5). Several sequences that hybridized with theOvIFNτ cDNA probe were identified. Several clones containing partialsequences of human interferon-τ were then isolated (Example 6). Twosynthetic 25-mer oligonucleotides, corresponding to sequences from theOvIFNτ cDNA (Imakawa, et al., 1987; Whaley, et al., 1994) weresynthesized. These primers were employed in amplification reactionsusing DNA derived from the following two cDNA libraries: human placentaand human cytotrophoblast cells isolated from term placenta. Theresulting amplified DNA fragments were electrophoretically separated anda band containing human IFNτ amplification products was isolated. Theamplification products were subcloned and the inserted amplificationproducts sequenced using the dideoxy termination method.

[0236] Comparison of sequences from five of these clones revealed a highdegree of sequence homology between the isolates, but the sequences werenot identical. This result suggests the existence of multiple variantsof human interferon-τ genes. Analysis of the nucleotide and proteinsequences suggests that human interferon-τ genes may be classified onthe basis of sequence homology into at least three groups. The groupsare presented below.

[0237] Example 7 describes the isolation of several full-length humanIFNτ genes. High molecular weight DNA was isolated from human peripheralblood mononuclear cells (PBMCs) and size-fractionated. Fractions weretested for the presence of IFNτ sequences using polymerase chainreaction: DNA molecules from fractions that tested amplificationpositive were used to generate a subgenomic library in gt11.

[0238] This subgenomic library was plated and hybridized with an OvIFNτcDNA probe (Example 7A). Approximately 20 clones were identified thathybridized to the probe. Plaques corresponding to the positive cloneswere passaged, DNA isolated and analyzed by amplification reactionsusing OvIFNτ primers. Of these twenty plaques, six plaques generatedpositive PCR signals. The phage from these six clones were purified andthe inserts sequenced. One of the inserts from one of these six cloneswas used as a hybridization probe in the following screening.

[0239] Recombinant phage from the λgt11 subgenomic library were screenedusing the hybridization probe just described (Example 7B). Five clonesgiving positive hybridization signals were isolated and the insertssequenced. The sequences from three of the clones overlapped, and theresulting consensus nucleic acid sequence (HuIFNτ1) is presented as SEQID NO:11 with the predicted protein coding sequence presented as SEQ IDNO:12. The predicted mature protein coding sequence is presented as SEQID NO:4. The sequences from the other two clones are presented as SEQ IDNO:29 (HuIFNτ2) and SEQ ID NO:31 (HuIFNτ3). The predicted mature aminoacid sequence from HuIFNτ2 is presented as SEQ ID NO:30. The predictedamino acid sequence from HuIFNτ3 is presented as SEQ ID NO:32, and themature amino acid sequence as SEQ ID NO:34.

[0240] Comparison of the predicted protein sequences (FIG. 3) of one ofthe human interferon-τ genes (SEQ ID NO:4) and the ovine interferon-τgene demonstrates the levels of sequence homology and divergence at theamino acid level.

[0241] An alignment of the nucleic acid sequences of the seven humaninterferon-τ nucleic acid sequences, described herein (Examples 6 and7), with ovine interferon-τ is shown in FIGS. 19A and 19B. Sequences ofOvIFNτ (oIFNτ), HuIFNτ1, HuIFNτ2, and HuIFNτ3 start at the upper leftcorner of FIG. 19A with the initiation ATG codon and continue throughthe second page of the figure. Sequences of HuIFNτ4, HuIFNτ5, HuIFNτ6and HuIFNτ7 start approximately half-way down FIG. 19A with the CAGcodon at amino acid position 40 (to the right of the exclamation marks)and continue through the second page of the figure. The 5′ and 3′ endsof each of the clones for HuIFNτ4, HuIFNτ5, HuIFNτ6 and HuIFNτ7 arerepresented by exclamation marks.

[0242] The complete coding sequence of OvIFNτ is presented in the toprow of each aligned set. Nucleotides in the other sequences areindicated only at positions where they differ from those of OvIFNτ.Lower case letters represent nucleotide changes that do not result inamino acid changes, while upper case letters represent those changesthat result in an amino acid substitution.

[0243] An alignment of the seven corresponding amino acid sequences,constructed in essentially the same manner as described above, ispresented in FIGS. 20A and 20B. As above, the complete amino acidsequence of OvIFNτ is presented in the top row, and amino acids of othersequences are indicated only at positions where they differ from theovine sequence.

[0244] An examination of the alignments reveals that the seven sequencesmay be grouped into at least three groups. Group I contains HuIFNτ1 andHuIFNτ2, group II contains HuIFNτ3, HuIFNτ4 and HuIFNτ5, and group IIIcontains HuIFNτ6 and HuIFNτ7. These groups may represent families ofinterferon-τ genes having distinct cellular functions.

[0245] These groupings were established based on the following criteria.In mature proteins, Group I HuIFNτs have an asparagine (ASN) at aminoacid position number 95 (numbers in reference to FIGS. 20A to 20B), amethionine (MET) at amino acid position number 104, and a leucine (LEU)at amino acid position number 120; Group II HuIFNτs have an asparticacid (ASP) at amino acid position number 95, a threonine (THR) at aminoacid position number 104, and a methionine (MET) at amino acid positionnumber 120; and Group III HuIFNτs have an arginine (ARG) at amino acidposition number 72, a valine (VAL) at amino acid position number 120,and a serine (SER) at amino acid position number 122.

[0246] The nucleic acid and polypeptide human IFNτ sequences presentedas SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34 can be used as the sourcefor specific primers and probes to detect isolates of further human IFNτcoding sequences and/or pseudogenes. Further, as described above, theremay be more than one isoform of the IFNτ protein and more than onecoding sequence per species. The specific nucleic acid probes used inthe practice of the present invention and antibodies reactive with theIFNτ polypeptides of the present invention may be useful to isolateunidentified variants of interferon-τ in mammals, according to themethods of the invention disclosed herein.

[0247] 2. Characterization of the Expression of Interferon-τ in HumanTissues.

[0248] Human placental cDNA libraries and an ovine cDNA library wereanalyzed by hybridization to the OvIFNτ cDNA probe (Example 8). This DNAhybridization analysis suggested that the IFNτ-signals from human cDNAlibraries were approximately 1/100 of the signal obtained using theovine cDNA library. OvIFNτ cDNAs constitute around 0.4% of the ovinecDNA library. Accordingly, the abundance of human cDNAs responding tothe OvIFNτ probe appears to be low, at least in the term placenta fromwhich the cDNA libraries were generated.

[0249] The presence of HuIFNτ mRNA in human term placenta and amniocyteswere also analyzed. The results suggest the presence of HuIFNτ mRNA inthe feto-placental annex. The aminocytes also expressed the messagescorresponding to OvIFNτ primers and human probe, suggesting that theexpression of IFNτ mRNA is not limited to the term placenta.

[0250] In addition, a RT-PCR analysis for the presence of HuIFNτ wasapplied to the total cellular RNA isolated from human adult lymphocytes:the results suggest that IFNτ mRNA exists in lymphocytes.

[0251] The expression of interferon-τ in human tissue was also examinedusing in situ hybridization (Example 9). Sections from four healthy,different term and first trimester human placentas were examined. Thisanalysis employed a cDNA probe derived from the OvIFNτ cDNA sequences(Example 9B). In situ hybridization was performed using an anti-senseRNA probe. In three separate experiments, specific hybridization wasobserved in all term and first trimester placental tissues.

[0252] First trimester placental villi (composed of an outer layer ofsyncytiotrophoblast, an underlying layer of cytotrophoblast, and acentral stromal region with various types of mesenchymal cells)displayed the highest transcript level of IFNτ in the cytotrophoblastcells. Less intense but detectable levels were present in both thesyncytiotrophoblast and stromal cells. A similar pattern of transcriptexpression was demonstrated in the placental villi of term tissue butthe level of signal detection was low. First trimester extravilloustrophoblasts displayed the highest amount of message and stainedpositive when present in the maternal blood spaces within the decidua.

[0253] Howatson, et al., (1988) noted IFNα production in thesyncytiotrophoblast of chorionic villi in both first trimester and termtissues. Also, Paulesu, et al. (1991) observed IFNα in extravilloustrophoblast as well as syncytiotrophoblasts, noting more intense andabundant reactivity in first trimester placental tissue when compared tothose taken at term. These investigators employed antibodies raisedagainst human IFNα subtypes, and none observed any IFNα in the villouscytotrophoblasts.

[0254] The present results demonstrate that the human IFNτ gene ishighly expressed in early placental tissues by migrating extravilloustrophoblasts, but is also expressed in villous syncytiotrophoblasts,cytotrophoblasts, and various stromal cells. These results demonstratethe detection of IFNτ transcripts in human pregnancy tissues, and IFNτexpression in the villous cytotrophoblasts as well as the extravilloustrophoblast of first trimester placenta.

[0255] C. Antiviral Properties of Interferon-τ.

[0256] The antiviral activity of OvIFNτ has been evaluated against anumber of viruses, including both RNA and DNA viruses. The relativespecific activity of OvIFNτ, purified to homogeneity, was evaluated inantiviral assays (Example 10). OvIFNτ had a higher specific antiviralactivity than either rBoIFNα or rBoIFNγ (Example 10, Table 3).

[0257] One advantage of the present invention is that OvIFNτ has potentantiviral activity with limited cytotoxic effects. Highly purifiedOvIFNτ was tested for anti-retroviral and cytotoxic effects onperipheral blood lymphocytes exposed to feline AIDS and human AIDSretroviruses (Bazer, F. W., et al., 1989). The feline AIDS lentivirusproduces a chronic AIDS-like syndrome in cats and is a model for humanAIDS (Pederson, et al., 1987). Replication of either virus in peripheralblood lymphocytes (PBL) was monitored by reverse transcriptase (RT)activity in culture supernatants over time.

[0258] To determine IFNτ antiviral activity against FIV and HIV,RNA-dependent DNA polymerase RT activity was assayed in FIV- andHIV-infected feline and human PBL cultures treated with OvIFNτ (Example11). Replication of FIV was reduced to about one-third of control valueswhen cells were cultured in the presence of OvIFNτ. Addition of OvIFNτproduced a rapid, dose-dependent decrease in reverse transcriptase (RT)activity (Example 11, Table 4). While concentrations as low as 0.62ng/ml of IFNτ inhibited viral replication, much higher concentrations(40 ng/ml) having greater effects on RT-activity were without toxiceffects on the cells. The results suggest that replication of the felineimmunodeficiency virus was reduced significantly compared to controlvalues when cells were cultured in the presence of OvIFNτ.

[0259] IFNτ appeared to exert no cytotoxic effect on the cells hostingthe retrovirus. This was true even when IFNτ was present at 40 ng per mlof culture medium. This concentration of IFNτ is equivalent to about8,000 antiviral units of alpha interferon—when OvIFNτ and HuIFNα areeach assayed for their ability to protect Madin-Darby bovine kidneycells from lysis by vesicular stomatitis virus (lysis assay as describedby Pontzer, et al., 1988).

[0260] IFNτ was also tested for activity against HIV replication inhuman cells. Human peripheral lymphocytes, which had been infected withHIV were treated with varying concentrations of IFNτ (Example 12).Replication of HIV in peripheral blood lymphocytes was monitored byreverse transcriptase activity in culture supernatants over time. Over arange of concentrations of IFNτ produced significant anti-HIV effects(Example 12, Table 5). A concentration of only 10 ng/ml resulted in overa 50% reduction in RT activity after only six days. A concentration of500 ng/ml resulted in a 90% reduction in RT activity within 10 days.Further, there was no evidence of any cytotoxic effects attributable tothe administration of IFNτ (Example 12, Table 5).

[0261] Further, the antiviral effects of IFNτ against HIV were evaluatedby treating human PBMC cells with various amounts of either recombinantIFNτ or recombinant human IFNα at the time of infection with HIV(Example 18). The data from these experiments (Example 18, Table 11)support the conclusion that, at similar concentrations, IFNα and IFNτare effective in reducing the replication of HIV in human lymphocytes.However, treatment of cells with IFNα resulted in cytotoxicity, whereasno such cytotoxicity was observed with treatment using IFNτ, even whenIFNτ was used at much higher concentrations. No cytotoxicity wasobserved using IFNτ, even when IFNτ was used at 200 times the dosage ofinterferon-alpha II.

[0262] Both FIV and HIV reverse transcriptase themselves were unaffectedby IFNτ in the absence of PBL. Therefore, the antiviral activity is notdue to a direct effect on the viral RT.

[0263] Interferon-τ has also been shown to inhibit Hepatitis B Virus DNAreplication in hepatocytes (Example 18). A human cell line derived fromliver cells transfected with Hepatitis B Virus (HBV) was used to testthe antiviral effects of IFNτ. The cells were treated with both the IFNαand IFNτ over a range of concentrations. Both IFNα and IFNτ reduced DNAproduction by approximately two-fold compared to the no interferoncontrol.

[0264] To demonstrate that the effect of the interferons was specific tothe infecting virus and not the result of an effect on general cellmetabolism, the hepatocytes were examined for the effects of IFNα andIFNτ on hepatospecific mRNA production (Example 18). Two hepatocytespecific proteins, Apo E and Apo A1, were detected by hybridizationanalysis. There was no apparent reduction of mRNA production for eitherhepatospecific mRNA at concentrations up to 40,000 units/ml of eitherIFNα or IFNτ. Further, no evidence for hepatotoxicity with IFNτ was seenin this assay.

[0265] The effects of recombinant ovine interferon tau (roIFNτ) on ovinelentivirus replication (OvLV) were also evaluated. In vitro effects wereassayed by infecting goat synovial membrane cells with serial dilutionsof OvLV. The infected cells were treated daily with roIFNτ (0-2,500antiviral units/ml [AVU/ml]) for 6 to 12 days, and virus replication andcytopathic effect (CPE; e.g., as in Example 2) were evaluated.

[0266] Evaluation methods included viral growth curves, cellproliferation assay (e.g., as in Examples 13, 14 or 15), syncytiaformation assay (e.g., as in Nagy, et al., Dalgleish, et al.), andquantitation of proviral DNA by PCR and reverse transcriptase assay(Mullis, Mullis, et al.). A reduction (p<0.001) of OvLV titer and CPE(80-99%) was observed in the roIFNτ-treated cultures.

[0267] In vivo effects of roIFNτ were assayed by inoculating twenty-fournewborn lambs with 5×10⁶ TCID₅₀ of OvLV strain 85/34. Eleven of theselambs were treated with 10⁵-10⁶ AVU/Kg of roIFNτ once a day for 30 dayspost-inoculation (PI) and twice a week thereafter. Thirteen lambs wereused as controls. Virus titers in blood, as determined by an end pointdilution method, peaked at 4-6 weeks PI in both groups. OvLV titers inroIFNτ-treated lambs were reduced relative to control animals. Thelargest reduction, a 90% decrease in OvLV titer in treated animalsrelative to control animals (p<0.01), was obtained 4 weeks PI.

[0268] The OvLV studies described above indicate that recombinant ovIFNτcan significantly reduce OvLV replication, and suggest that IFNτ may beused to control clinical diseases caused by lentivirus infections. Takentogether with the other antiviral data, these results suggest that IFNτis an effective antiviral agent against a wide variety of viruses,including both RNA and DNA viruses.

[0269] Ovine interferon-τ may be useful in veterinary applicationsincluding, but not limited to, the treatment of the following viraldiseases: feline leukemia virus, ovine progressive pneumonia virus,ovine lentivirus, equine infectious anemia virus, bovineimmunodeficiency virus, visna-maedi virus, and caprine arthritisencephalitis.

[0270] Human interferon-τ may be used for the treatment of, for example,the following viral diseases: human immunodeficiency virus (HIV),hepatitis c virus (HCV) and hepatitis B virus (HBV).

[0271] D. Antiproliferative Properties of IFNτ.

[0272] The effects of IFNτ on cellular growth have also been examined.In one analysis, anti-cellular growth activity was examined using acolony inhibition assay (Example 13). Human amnion (WISH) or MDBK cellswere plated at low cell densities to form colonies originating fromsingle cells. Dilutions of interferons were added to triplicate wellsand the plates were incubated to allow colony formation. IFNτ inhibitedboth colony size and number in these assays. IFNτ was more effective atinhibiting cell proliferation of the human cell line (WISH) than humanIFNα. The antiproliferative activity of IFNτ was dose-dependent. Highconcentrations of IFNτ stopped proliferation, while cell viability wasnot impaired.

[0273] Based on cell cycle analysis, using flow cytometry, IFNτ appearsto inhibit progress of cells through S phase. These results demonstratethe antiproliferative effect of IFNτ, and underscore its lowcytotoxicity.

[0274] The antiproliferative effects of IFNτ were also studied for ratand bovine cell lines (Example 14). The rate of ³H-thymidineincorporation was used to assess the rate of cellular proliferation. Thedata obtained demonstrate that IFNτ drastically reduced the rate ofcellular proliferation (Example 14, Table 7) for each tested cell line.

[0275] The antiproliferative activity and lack of toxicity of IFNτ wasfurther examined using a series of human tumor cell lines (Example 15).A variety of human tumor cell lines were selected from the standardlines used in NIH screening procedure for antineoplastic agents(Pontzer, C. H., et al., (1991)). At least one cell line from each majorneoplastic category was examined.

[0276] The following cell lines were obtained from American Type CultureCollection (12301 Parklawn Dr., Rockville Md. 20852): NCI-H460 humanlung large cell carcinoma; DLD-1 human colon adenocarcinoma; SK-MEL-28human malignant melanoma; ACHN human renal adenocarcinoma; HL-60 humanpromyelocytic leukemia; H9 human T cell lymphoma; HUT 78 human cutaneousT cell lymphoma; and MCF7 human breast adenocarcinoma.

[0277] As above, the antiproliferative activity was evaluated bymeasuring the rate of ³H-thymidine incorporation into cells which havebeen treated with IFNτ. Significant differences between treatments wereassessed by an analysis of variance followed by Scheffe's F-test. Cellcycle analysis was performed by flow cytometry.

[0278] Examination of IFNτ inhibition of MCF7 (breast adenocarcinoma)proliferation demonstrated that IFNτ reduced MCF7 proliferation in adose-dependent manner. A 50% reduction in ³H-thymidine was observed with10,000 units/ml of IFNτ (Example 15, Table 8). This cell line hadpreviously been found to be unresponsive to anti-estrogen treatment.

[0279] A comparison of the antiproliferative effects of IFNτ and IFNαwas conducted using HL-60 (human promyelocytic leukemia) cells. Resultswith the promyelocytic leukemia HL-60 are typical of those obtainedcomparing IFNτ with human IFNα (Example 15). Concentrations as low as100 units/ml of both IFNs produced significant (>60%) growth reduction.Increasing amounts of IFNs further decreased tumor cell proliferation(FIG. 4). High doses of HuIFNα, but not OvIFNτ, were cytotoxic (FIG. 5).Cell viability was reduced by approximately 80% by IFNα. By contrast,nearly 100% of the IFNτ-treated cells remained viable when IFNτ wasapplied at 10,000 units/ml. Thus, while both interferons inhibitproliferation, only IFNτ is without cytotoxicity. This lack of toxicityprovides an advantage of IFNτ for use in vivo therapies.

[0280] The human cutaneous T cell lymphoma, HUT 78, responded similarlyto HL-60 when treated with IFNτ (Example 15, FIG. 9). Both OvIFNτ andrHuIFNα reduce HUT 78 cell growth, but IFNα demonstrated adverse effectson cell viability.

[0281] The T cell lymphoma H9 was less sensitive to theantiproliferative effects of IFNα than the tumor cell lines describedabove. While IFNα was not toxic to the H9 cells, it failed to inhibitcell division significantly at any of the concentrations examined(Example 15, FIG. 10). In contrast, IFNτ was observed to reduce H9growth by approximately 60%. Thus, only OvIFNτ is an effective growthinhibitor of this T cell lymphoma.

[0282] In three additional tumor cell lines (NCI-H460, DLD-1 andSK-MEL-28) IFNτ and IFNα were equally efficacious antitumor agents. Inthe melanoma, SK-MEL-28, inhibition of proliferation by IFNα wasaccomplished by a 13% drop in viability, while IFNτ was not cytotoxic.In the majority of tumors examined, IFNτ is equal or preferable to IFNαas an antineoplastic agent against human tumors.

[0283] IFNτ exhibits antiproliferative activity against human tumorcells without toxicity and is as potent or more potent than human IFNα.Clinical trials of the IFNα2s have shown them to be effective antitumoragents (Dianzani, F., 1992; Krown, 1987). One advantage of IFNτ as atherapeutic is the elimination of toxic effects seen with high dosesIFNαs.

[0284] An additional application of the IFNτ is against tumors likeKaposi's sarcoma (associated with HIV infection) where theantineoplastic effects of IFNτ are coupled with IFNτ ability to inhibitretroviral growth.

[0285] The in vivo efficacy of interferon-τ treatment was examined in amouse system (Example 16). B16-F10 is a syngeneic mouse transplantabletumor selected because of its high incidence of pulmonary metastases(Poste, et al., 1981). Interferon treatment was initiated 3 days afterthe introduction of the tumor cells. The in vivo administration of IFNτdramatically reduced B16-F10 pulmonary tumors. Thus, IFNτ appears to bean efficacious antineoplastic agent in vivo as well as in vitro.

[0286] These results support the usefulness of human IFNτ for use inmethods to inhibit or reduced tumor cell growth, including, but are notlimited to, the following types of tumor cells: human carcinoma cells,hematopoietic cancer cells, human leukemia cells, human lymphoma cells,human melanoma cells and steroid-sensitive tumor cells (for example,mammary tumor cells).

[0287] E. Cytotoxicity of Interferons.

[0288] One advantage of IFNτ over other interferons, such as IFNα, isthat treatment of a subject with therapeutic doses of IFNτ does notappear to be associated with cytotoxicity. In particular, IFN-τ appearsto be non-toxic at concentrations at which IFN-β induces toxicity. Thisis demonstrated by experiments in which L929 cells were treated withvarious concentrations of either oIFNτ or MuIFN-β (Lee Biomolecular, SanDiego, Calif.), ranging from 6000 U/ml to 200,000 U/ml (Example 18E).

[0289] oIFNτ, MuIFN-β or medium (control) were added at time zero andthe cells were incubated for 72 hours. The results of the experimentsare presented in FIG. 21. The percent of live cells (relative tocontrol) is indicated along the y-axis (±standard error). One hundredpercent is equal to the viability of L929 cells treated with mediumalone. The results indicate that oIFNτ is essentially non-toxic atconcentrations up to 100,000 U/ml, and is significantly less toxic thanMuIFN-β over the entire therapeutic range of the compounds.

[0290] It has been previously demonstrated that in vivo treatment withboth of the type I IFNs, IFNβ and IFNα in humans and animals causestoxicity manifested as a number of side effects including fever,lethargy, tachycardia, weight loss, and leukopenia (Degre, 1974; Fentand Zbinden, 1987). The effect of in vivo treatment with IFNτ, IFNβ andIFNα (10⁵ U/injection) on total white blood cell (WBC), total lymphocytecounts and weight measurements in NZW mice (Table 13) was examined asdescribed in Example 18F. No significant difference between IFNτ treatedand untreated mice was observed for WBC, lymphocyte counts or weightchange.

[0291] In comparison, IFNβ treated mice exhibited a 31.7% depression inlymphocyte counts 12 hours after injection. Further, depression oflymphocyte counts continued 24 hours after IFNβ injection. IFNα treatedmice exhibited a 55.8% lymphocyte depression and significant weight loss12 hours after injection. Thus, IFNτ appears to lack toxicity in vivounlike IFNβ and IFNα as evidenced by studies of peripheral blood andweight measurements.

[0292] oIFNτ is 172 amino acids long compared to 165 or 166 amino acidsfor IFNα. The carboxyl-terminal portion of oIFNτ is hydrophilic andthought to be surface accessible. To explore whether this carboxyl“tail” interacts with the binding epitope of oIFNτ to mediate therelative lack of cytotoxicity, a series of deletion mutants weregenerated.

[0293] The carboxyl terminal 2, 6 and 10 amino acids of oIFNτ wereremoved by cassette mutagenesis of a synthetic oIFNτ gene. The mutant(variant) synthetic genes were cloned into the pHIL-S1 Pichia expressionplasmid (Invitrogen, San Diego, Calif.), the plasmid was cut with BglII,and the linearized plasmid was used to transform Pichia pastoris (strainGS115; Invitrogen) spheroplasts according to the manufacturer'sinstructions.

[0294] Recombinant variant proteins expressed by transformed His⁺ Mut⁻yeast cells were purified and used to determine in vitro antiviralactivity and relative cytotoxicity of the variants compared to intactoIFNτ and IFN-α. The cytoxicity of the variants was distributed betweenthe that of oIFNτ and IFN-α. Variants with shorter deletions were moresimilar in their cytotoxic properties to oIFNτ, while those with longerdeletions were more similar to IFN-α.

[0295] While not wishing to be bound by any specific molecularmechanisms underlying the properties of IFNτ, the results of theexperiments suggest that the C-terminus 10 amino acids of IFNτ may playa role in the decreased cytotoxicity of IFNτ relative to otherinterferons.

[0296] III. Interferon-τ Polypeptide Fragments, Protein Modeling andProtein Modifications.

[0297] A. IFNτ Polypeptide Fragments.

[0298] The variety of IFNτ activities, its potency and lack ofcytotoxicity, as taught by the present specification, suggest theimportance of structure/function analysis for this novel interferon. Thestructural basis for OvIFNτ function has been examined using sixoverlapping synthetic peptides corresponding to the entire OvIFNτsequence (FIG. 6). The corresponding polypeptides derived from the ovineIFNτ sequence are presented as SEQ ID NO:5 to SEQ ID NO:10. Threepeptides representing amino acids 1-37, 62-92 and 139-172 have beenshown to inhibit IFNτ antiviral activity (Example 17). The peptides wereeffective competitors at concentrations of 300 μM and above.

[0299] The synthetic polypeptide representing the C-terminal region ofovIFNτ, OvIFNτ (139-172), and the internal peptide OvIFNτ (62-92),inhibited IFNτ and rBoIFNα_(II) antiviral activity to the same extent,while the N-terminal peptide OvIFNτ (1-37) was more effective ininhibiting OvIFNτ antiviral activity. Dose-response data indicated thatIFNτ (62-92) and IFNτ (139-172) inhibited IFNτ antiviral activity tosimilar extents. The same peptides that blocked IFNτ antiviral activityalso blocked the antiviral activity of recombinant bovine IFNα(rBoIFNα); recombinant bovine IFNγ was unaffected by the peptides. Thesetwo IFNτ peptides may represent common receptor binding regions for IFNτand various IFNαs.

[0300] The two synthetic peptides OvIFNτ (1-37) and OvIFNτ (139-172)also blocked OvIFNτ anti-FIV and anti-HIV activity (Example 17; FIGS.11A and 11B). While both peptides blocked FIV RT activity, only theC-terminal peptide, OvIFNτ (139-172), appeared to be an efficientinhibitor of vesicular stomatitis virus activity on the feline cellline, Fc9.

[0301] The above data taken together suggest that the C-terminal regionsof type I interferons may bind to common site on the type I interferonreceptor, while the N-terminal region may be involved in the elicitationof unique functions. These results suggest that portions of the IFNτmolecule may be used to substitute regions of interferon alphamolecules. For example, the region of an interferon alpha molecule thatis responsible for increased cytotoxicity, relative to IFNτ treatment,can be identified by substituting polypeptide regions derived from IFNτfor regions of an interferon alpha molecule. Such substitutions can becarried out by manipulation of synthetic genes (see below) encoding theselected IFNτ and interferon alpha molecules, coupled to the functionalassays described herein (such as, antiviral, antiproliferative andcytoxicity assays).

[0302] Polyclonal anti-peptide antisera against the IFNτ peptidesyielded similar results as the polypeptide inhibition studies, describedabove. Antibodies directed against the same three regions (OvIFNτ(1-37), IFNτ (62-92) and IFNτ (139-172)) blocked OvIFNτ function,confirming the importance of these three domains in antiviral activity(Example 17). These peptides, although apparently binding to theinterferon receptor; did not in and of themselves elicit interferon-likeeffects in the cells.

[0303] The antiproliferative activity of IFNτ (Example 17, Table 11)involved a further region of the molecule, since IFNτ (119-150) was themost effective inhibitor of OvIFNτ-induced reduction of cellproliferation. This results suggests that the region of the moleculeprimarily responsible for inhibition of cell growth is the IFNτ(119-150) region. This region of the IFNτ molecule may be useful aloneor fused to other proteins (such as serum albumin, an antibody or aninterferon alpha polypeptide) as an antineoplastic agent. A conjugatedprotein between an N-terminal peptide derived from human interferon-αand serum albumin was shown to have anticellular proliferation activity(Ruegg, et al., 1990).

[0304] Finally, binding of ¹²⁵I-OvIFNτ to its receptor on MDBK cellscould be blocked by antisera to 4 of the 6 peptides; the 4 polypeptidesrepresenting amino acids 1-37, 62-92, 119-150 and 139-172 of OvIFNτ.This reflects the multiple binding domains as well as the functionalsignificance of these regions. Since different regions of IFNτ areinvolved in elicitation of different functions, modification of selectedamino acids could potentially result in IFNτ-like interferons withselective biological activity.

[0305] Polypeptide fragments of human IFNτ proteins, having similarproperties to the OvIFNτ polypeptides just described, are proposed basedon the data presented above for OvIFNτ polypeptide fragments combinedwith the HuIFNτ sequence information disclosed herein. Suchhuman-sequence derived polypeptides include, but are not limited to, thefollowing: SEQ ID NO:15 to SEQ ID NO:20, and SEQ ID NO:35 to SEQ IDNO:40.

[0306] The above data demonstrate the identification of syntheticpeptides having four discontinuous sites on the OvIFNτ protein that areinvolved in receptor interaction and biological activity. In order toelucidate the structural relationship of these regions, modeling of thethree dimensional structure of IFNτ was undertaken. A three dimensionalmodel would be useful in interpretation of existing data and the designof future structure/function studies.

[0307] B. Molecular Modeling

[0308] Combining circular dichroism (CD) data of both the full lengthrecombinant OvIFNτ and IFNβ (a protein of known three dimensionalstructure (Senda, et al., 1992)), a model of OvIFNτ was constructed. Themost striking feature of this model is that IFNτ falls into a class ofproteins with a four-helix bundle motif. The CD spectra of IFNτ wastaken on an AVIV 60 S spectropolarimeter. Two different methods wereemployed for secondary structure estimations, the algorithm of Perczel,et al., (1991) and variable selection by W. C. Johnson, Jr. (1992).

[0309] Secondary structure estimations of the spectra indicate 70-75%alpha helix (characterized by minima at 222 and 208 nm and maximum at190 nm). The variable selection algorithm estimates the remainder of themolecule to be 20% beta sheet and 10% turn. The Chang method estimatesthe remainder to be 30% random coil. Alignment of IFNτ and IFNβsequences revealed homology between the two molecules, specifically inthe regions of known helical structure in IFNβ. Sequence analysis ofIFNτ also showed that proposed helical regions possess an apolarperiodicity indicative of a four-helix bundle motif.

[0310] The final modeling step was to apply the IFNβ x-raycrystallographic coordinates of the IFNβ carbon backbone to the IFNτsequence. The functionally active domains of IFNτ, identified above,were localized to one side of the molecule and found to be in closespatial proximity. This is consistent with multiple binding sites onIFNτ interacting simultaneously with the type I IFN receptor.

[0311] The three dimensional modeling data coupled with the functiondata described above, provides the information necessary to introducesequence variations into specific regions of IFNτ to enhance selectedfunctions (e.g., antiviral or anticellular proliferation) or tosubstitute a region(s) of selected function into other interferonmolecules (e.g., antiviral, antineoplastic, or reduced cytotoxicity).

[0312] C. Recombinant and Synthetic Manipulations

[0313] The construction of a synthetic gene for OvIFNτ is described inExample 3. Briefly, an amino acid sequence of ovIFNτ was back-translatedfrom an ovIFNτ cDNA (Imakawa, et al., 1987) using optimal codon usagefor E. coli. The sequence was edited to include 20, unique, restrictionsites spaced throughout the length of the construct. This 540 base pairsynthetic gene sequence was divided into 11 oligonucleotide fragments.Individual fragments were synthesized and cloned, either single ordouble stranded, into either pTZ 19R, pTZ 18R or pBluescript, amplifiedand fused. The synthetic OvIFNτ construct was then cloned into amodified pIN-III-ompA expression vector for expression in bacteria andalso cloned into a yeast expression plasmid. A similarly constructedhuman IFNτ synthetic gene (SEQ ID NO:3) has been designed, constructedand expressed in yeast cells.

[0314] Expression of the OvIFNτ synthetic gene in yeast (Example 4)allowed over production of recombinant IFNτ in S. cerevisiae: largequantities (5-20 mg/l) of recombinant IFNτ can be purified from solubleyeast extract using sequential ion exchange and molecular sievechromatography. Recombinant IFNτ purified in this fashion exhibitedpotent antiviral activity (2 to 3×10⁸ units/mg) similar to nativeOvIFNτ.

[0315] The synthetic gene construct facilitates introduction ofmutations for possible enhancement of antitumor (anticellularproliferative) and antiviral activities. Further, the disparate regionsof the molecule responsible for different functions can be modifiedindependently to generate a molecule with a desired function. Forexample, two deletion mutants, OvIFNτ (1-162) and OvIFNτ (1-166), havebeen constructed to examine the role of carboxy terminal sequences inIFNτ molecules.

[0316] Additional mutant IFNτ molecules have been constructed toidentify residues critical for antiproliferative activity. For example,one particular residue, TYR 123 has been implicated in the anticellularproliferative activity of IFNα (McInnes, et al., 1989). The equivalentof TYR 123 in IFNτ is contained within peptide OvIFNτ (119-150): thispolypeptide inhibits OvIFNτ and human IFNα antiproliferative activity.Mutations converting TYR 123 to conservative (TRP) and nonconservative(ASP) substitutions have been generated, as well as mutant sequenceshaving deletion of this residue. The codon for TYR 123 is located withinan SspI site; elimination of this site has been used for screening. Theantiproliferative activity of these mutant IFNτ is evaluated asdescribed herein.

[0317] Synthetic peptides can be generated which correspond to the IFNτpolypeptides of the present invention. Synthetic peptides can becommercially synthesized or prepared using standard methods andapparatus in the art (Applied Biosystems, Foster City Calif.).

[0318] Alternatively, oligonucleotide sequences encoding peptides can beeither synthesized directly by standard methods of oligonucleotidesynthesis, or, in the case of large coding sequences, synthesized by aseries of cloning steps involving a tandem array of multipleoligonucleotide fragments corresponding to the coding sequence (Crea;Yoshio et al.; Eaton et al.). Oligonucleotide coding sequences can beexpressed by standard recombinant procedures (Maniatis et al.; Ausubelet al.).

[0319] The biological activities of the interferon-τ polypeptidesdescribed above can be exploited using either the interferon-τpolypeptides alone or conjugated with other proteins (see below).

[0320] IV. Production of Fusion Proteins.

[0321] In another aspect, the present invention includes interferon-τ orinterferon-τ-derived polypeptides covalently attached to a secondpolypeptide to form a fused, or hybrid, protein. The interferon-τsequences making up such fused proteins can be recombinantly producedinterferon-τ or a bioactive portion thereof, as described above.

[0322] For example, where interferon-τ is used to inhibit viralexpression, polypeptides derived from IFNτ demonstrating antiviralactivity may be advantageously fused with a soluble peptide, such as,serum albumin, an antibody (e.g., specific against an virus-specificcell surface antigen), or an interferon alpha polypeptide. In oneembodiment, the IFNτ polypeptides provide a method of reducing thetoxicity of other interferon molecules (e.g., IFNβ or IFNα) by replacingtoxicity-associated regions of such interferons with, for example,corresponding interferon-τ regions having lower toxicity. In anotherembodiment, fusion proteins are generated containing interferon-τregions that have anticellular proliferation properties. Such regionsmay be obtained from, for example, the interferon-τ sequences disclosedherein. Other examples of fusion proteins include (i) replacingtoxicity-associated regions of interferon-α with the interferon-τregions SEQ ID NO:5 and SEQ ID NO:15, and (ii) fusion proteinscontaining the interferon-τ regions SEQ ID NO:9 and SEQ ID NO:19 asanticellular proliferation agents.

[0323] The fused proteins of the present invention may be formed bychemical conjugation or by recombinant techniques. In the former method,the interferon-τ and second selected polypeptide are modified byconventional coupling agents for covalent attachment. In one exemplarymethod for coupling soluble serum albumin to an interferon-τpolypeptide, serum albumin is derivatized with N-succinimidyl-S-acetylthioacetate (Duncan), yielding thiolated serum albumin. The activatedserum albumin polypeptide is then reacted with interferon-τ derivatizedwith N-succinimidyl 3-(2-pyridyldithio) propionate (Cumber), to producethe fused protein joined through a disulfide linkage.

[0324] As an alternative method, recombinant interferon-τ may beprepared with a cysteine residue to allow disulfide coupling of theinterferon-τ to an activated ligand, thus simplifying the couplingreaction. An interferon-τ expression vector, used for production ofrecombinant interferon-τ, can be modified for insertion of an internalor a terminal cysteine codon according to standard methods ofsite-directed mutagenesis (Ausubel, et al.).

[0325] In one method, a fused protein is prepared recombinantly using anexpression vector in which the coding sequence of a second selectedpolypeptide is joined to the interferon-τ coding sequence. For example,human serum albumin coding sequences can be fused in-frame to the codingsequence of an interferon-τ polypeptide, such as, SEQ ID NO:9, SEQ IDNO:19 or SEQ ID NO:39.

[0326] The fused protein is then expressed using a suitable host cell.

[0327] The fusion protein may be purified by molecular-sieve andion-exchange chromatography methods, with additional purification bypolyacrylamide gel electrophoretic separation and/or HPLCchromatography, if necessary.

[0328] It will be appreciated from the above how interferon-τ-containingfusion proteins may be prepared. One variation on the above fusion is toexchange positions of the interferon-τ and selected second proteinmolecules in the fusion protein (e.g., carboxy terminal versus aminoterminal fusions). Further, internal portions of a native interferon-τpolypeptide (for example, amino acid regions of between 15 and 172 aminoacids) can be assembled into polypeptides where two or more suchinterferon-τ portions are contiguous that are normally discontinuous inthe native protein.

[0329] In addition to the above-described fusion proteins, the presentinvention also contemplates polypeptide compositions having (a) a humaninterferon-τ polypeptide, where said polypeptide is (i) derived from aninterferon-τ amino acid coding sequence, and (ii) between 15 and 172amino acids long, and (b) a second soluble polypeptide. Interferon-a andinterferon-β are examples of such second soluble polypeptides. IFNτpolypeptides associated with reduced toxicity may be co-administeredwith more toxic interferons to reduce the toxicity of the more toxicinterferons when used in pharmaceutical formulations or in therapeuticapplications. Such IFNτ polypeptides would, for example, reduce thetoxicity of IFNα but not interfere with IFNα antiviral properties.

[0330] V. Antibodies Reactive with Interferon-τ.

[0331] Fusion proteins containing the polypeptide antigens of thepresent invention fused with the glutathione-S-transferase (Sj26)protein can be expressed using the pGEX-GLI vector system in E. coliJM101 cells. The fused Sj26 protein can be isolated readily byglutathione substrate affinity chromatography (Smith). Expression andpartial purification of IFNτ proteins is described in (Example 20), andis applicable to any of the other soluble, induced polypeptides coded bysequences described by the present invention.

[0332] Insoluble GST (sj26) fusion proteins can be purified bypreparative gel electrophoresis.

[0333] Alternatively, IFNτ-β-galactosidase fusion proteins can beisolated as described in Example 19.

[0334] Also included in the invention is an expression vector, such asthe lambda gt11 or pGEX vectors described above, containing IFNτ codingsequences and expression control elements which allow expression of thecoding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector.

[0335] The DNA encoding the desired polypeptide can be cloned into anynumber of vectors (discussed above) to generate expression of thepolypeptide in the appropriate host system. These recombinantpolypeptides can be expressed as fusion proteins or as native proteins.A number of features can be engineered into the expression vectors, suchas leader sequences which promote the secretion of the expressedsequences into culture medium. Recombinantly produced IFNτ, andpolypeptides derived therefrom, are typically isolated from lysed cellsor culture media. Purification can be carried out by methods known inthe art including salt fractionation, ion exchange chromatography, andaffinity chromatography. Immunoaffinity chromatography can be employedusing antibodies generated against selected IFNτ antigens.

[0336] In another aspect, the invention includes specific antibodiesdirected against the polypeptides of the present invention. Typically,to prepare antibodies, a host animal, such as a rabbit, is immunizedwith the purified antigen or fused protein antigen. Hybrid, or fused,proteins may be generated using a variety of coding sequences derivedfrom other proteins, such as β-galactosidase orglutathione-S-transferase. The host serum or plasma is collectedfollowing an appropriate time interval, and this serum is tested forantibodies specific against the antigen. Example 20 describes theproduction of rabbit serum antibodies which are specific against theIFNτ antigens in a Sj26/IFNτ hybrid protein. These techniques can beapplied to the all of the IFNτ molecules and polypeptides derivedtherefrom.

[0337] The gamma globulin fraction or the IgG antibodies of immunizedanimals can be obtained, for example, by use of saturated ammoniumsulfate or DEAE Sephadex, or other techniques known to those skilled inthe art for producing polyclonal antibodies.

[0338] Alternatively, purified protein or fused protein may be used forproducing monoclonal antibodies. Here the spleen or lymphocytes from aanimal immunized with the selected polypeptide antigen are removed andimmortalized or used to prepare hybridomas by methods known to thoseskilled in the art (Harlow, et al.). Lymphocytes can be isolated from aperipheral blood sample. Epstein-Barr virus (EBV) can be used toimmortalize human lymphocytes or a fusion partner can be used to producehybridomas.

[0339] Antibodies secreted by the immortalized cells are screened todetermine the clones that secrete antibodies of the desired specificity,for example, by using the ELISA or Western blot method (Ausubel et al.).Experiments performed in support of the present invention have yieldedfour hybridomas producing monoclonal antibodies specific for ovine IFNτhave been isolated.

[0340] Antigenic regions of polypeptides are generally relatively small,typically 7 to 10 amino acids in length. Smaller fragments have beenidentified as antigenic regions. Interferon-τ polypeptide antigens areidentified as described above. The resulting DNA coding regions can beexpressed recombinantly either as fusion proteins or isolatedpolypeptides.

[0341] In addition, some amino acid sequences can be convenientlychemically synthesized (Applied Biosystems, Foster City Calif.).Antigens obtained by any of these methods may be directly used for thegeneration of antibodies or they may be coupled to appropriate carriermolecules. Many such carriers are known in the art and are commerciallyavailable (e.g., Pierce, Rockford Ill.).

[0342] Antibodies reactive with IFNτ are useful, for example, in theanalysis of structure/function relationships.

[0343] VI. Utility

[0344] A. Reproductive.

[0345] Although IFNτ bears some similarity to the IFNα family based onstructure and its potent antiviral properties, the IFNαs do not possessthe reproductive properties associated with IFNτ. For example,recombinant human IFNα had no effect on interestrous interval comparedto IFNτ, even when administered at twice the dose (Davis, et al., 1992).

[0346] Therefore, although IFNτ has some structural similarities toother interferons, it has very distinctive properties of its own: forexample, the capability of significantly influencing the biochemicalevents of the estrous cycle.

[0347] The human IFNτ of the present invention can be used in methods ofenhancing fertility and prolonging the life span of the corpus luteum infemale mammals as generally described in Hansen, et al., hereinincorporated by reference. Further, the human interferon-τ of thepresent invention could be used to regulate growth and development ofuterine and/or fetal-placental tissues. The human IFNτ is particularlyuseful for treatment of humans, since potential antigenic responses areless likely using such a same-species protein.

[0348] B. Antiviral Properties.

[0349] The antiviral activity of IFNτ has broad therapeutic applicationswithout the toxic effects that are usually associated with IFNαs.Although the presence of IFNτ in culture medium inhibited reversetranscriptase activity of the feline immunodeficiency virus (Example11), this is not due to a direct effect of IFNτ on the reversetranscriptase. Rather, IFNτ appears to induce the host cell to produce afactor(s) which is inhibitory to the reverse transcriptase of the virus.

[0350] IFNτ was found to exert its antiviral activity without adverseeffects on the cells: no evidence of cytotoxic effects attributable tothe administration of IFNτ was observed. It is the lack of cytotoxicityof IFNτ which makes it extremely valuable as an in vivo therapeuticagent. This lack of cytotoxicity sets IFNτ apart from most other knownantiviral agents and all other known interferons.

[0351] Formulations comprising the IFNτ compounds of the presentinvention can be used to inhibit viral replication.

[0352] The human IFNτ of the present invention can be employed inmethods for affecting the immune relationship between fetus and mother,for example, in preventing transmission of maternal viruses (e.g., HIV)to the developing fetus. The human interferon-τ is particularly usefulfor treatment of humans, since potential antigenic responses are lesslikely using a homologous protein.

[0353] C. Anticellular Proliferation Properties.

[0354] IFNτ exhibits potent anticellular proliferation activity. IFNτcan also be used to inhibit cellular growth without the negative sideeffects associated with other interferons which are currently known.Formulations comprising the IFNτ compounds of the subject invention canbe used to inhibit, prevent, or slow tumor growth.

[0355] The development of certain tumors is mediated by estrogen.Experiments performed in support of the present invention indicate thatIFNτ can suppress estrogen receptor numbers. Therefore, IFNτ can be usedin the treatment or prevention of estrogen-dependent tumors.

[0356] D. Interfering with the Binding of Interferons to Receptors.

[0357] IFNτ appears to interact with the Type I IFN receptor via severalepitopes on the molecule, and these regions either separately or incombination may affect distinct functions of IFNτ differently.

[0358] The polypeptides of the present invention are useful for theselective inhibition of binding of interferons to the interferonreceptor. Specifically, as described herein, certain of the disclosedpeptides selectively inhibit the antiviral activity of IFNτ while othersinhibit the antiproliferative activity. Combinations of these peptidescould be used to inhibit both activities. Advantageously, despitebinding to the interferon receptor and blocking IFNτ activity, thesepeptides do not, themselves, elicit the antiviral or antiproliferativeactivity.

[0359] Therefore, such polypeptides can be used as immunoregulatorymolecules when it is desired to prevent immune responses triggered byinterferon molecules. These peptides could be used as immunosuppressantsto prevent, for example, interferon-mediated immune responses to tissuetransplants. Other types of interferon mediated responses may also beblocked, such as the cytotoxic effects of alpha interferon.

[0360] E. Pharmaceutical Compositions.

[0361] IFNτ proteins can be formulated according to known methods forpreparing pharmaceutically useful compositions. Formulations comprisinginterferons or interferon-like compounds have been previously described(for example, Martin, 1976). In general, the compositions of the subjectinvention will be formulated such that an effective amount of the IFNτis combined with a suitable carrier in order to facilitate effectiveadministration of the composition.

[0362] The compositions used in these therapies may also be in a varietyof forms. These include, for example, solid, semi-solid, and liquiddosage forms, such as tablets, pills, powders, liquid solutions orsuspensions, liposomes, suppositories, injectable, and infusiblesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions alsopreferably include conventional pharmaceutically acceptable carriers andadjuvants which are known to those of skill in the art. Preferably, thecompositions of the invention are in the form of a unit dose and willusually be administered to the patient one or more times a day.

[0363] IFNτ, or related polypeptides, may be administered to a patientin any pharmaceutically acceptable dosage form, including oral intake,inhalation, intranasal spray, intraperitoneal, intravenous,intramuscular, intralesional, or subcutaneous injection. Specifically,compositions and methods used for other interferon compounds can be usedfor the delivery of these compounds.

[0364] One primary advantage of the compounds of the subject invention,however, is the extremely low cytotoxicity of the IFNτ proteins. Becauseof this low cytotoxicity, it is possible to administer the IFNτ inconcentrations which are greater than those which can generally beutilized for other interferon (e.g., IFNα) compounds. Thus, IFNτ can beadministered at rates from about 5×10⁴ to 20×10⁶ units/day to about500×10⁶ units/day or more. In a preferred embodiment, the dosage isabout 20×10⁶ units/day. High doses are preferred for systemicadministration. It should, of course, be understood that thecompositions and methods of this invention may be used in combinationwith other therapies.

[0365] Once improvement of a patient's condition has occurred, amaintenance dose is administered if necessary. Subsequently, the dosageor the frequency of administration, or both, may be reduced, as afunction of the symptoms, to a level at which the improved condition isretained. When the symptoms have been alleviated to the desired level,treatment should cease. Patients may, however, require intermittenttreatment on a long-term basis upon any recurrence of disease symptoms.

[0366] The compositions of the subject invention can be administeredthrough standard procedures to treat a variety of cancers and viraldiseases including those for which other interferons have previouslyshown activity. See, for example, Finter, et al. (1991); Dianzani, etal. (1992); Francis, et al. (1992) and U.S. Pat. Nos. 4,885,166 and4,975,276. However, as discussed above, the compositions of the subjectinvention have unique features and advantages, including their abilityto treat these conditions without toxicity.

[0367] F. Treatment of Skin Disorders.

[0368] Disorders of the skin can be treated intralesionally using IFNτ,wherein formulation and dose will depend on the method of administrationand on the size and severity of the lesion to be treated. Preferredmethods include intradermal and subcutaneous injection. Multipleinjections into large lesions may be possible, and several lesions onthe skin of a single patient may be treated at one time. The schedulefor administration can be determined by a person skilled in the art.Formulations designed for sustained release can reduce the frequency ofadministration.

[0369] G. Systemic Treatment.

[0370] Systemic treatment is essentially equivalent for allapplications. Multiple intravenous, subcutaneous and/or intramusculardoses are possible, and in the case of implantable methods fortreatment, formulations designed for sustained release are particularlyuseful. Patients may also be treated using implantable subcutaneousportals, reservoirs, or pumps.

[0371] H. Regional Treatment.

[0372] Regional treatment with the IFNτ polypeptides of the presentinvention is useful for treatment of cancers in specific organs.Treatment can be accomplished by intraarterial infusion. A catheter canbe surgically or angiographically implanted to direct treatment to theaffected organ. A subcutaneous portal, connected to the catheter, can beused for chronic treatment, or an implantable, refillable pump may alsobe employed.

[0373] The following examples illustrate, but in no way are intended tolimit the present invention.

Materials and Methods

[0374] Restriction endonucleases, T4 DNA ligase, T4 polynucleotidekinase, Taq DNA polymerase, and calf intestinal phosphatase werepurchased from New England Biolabs (Beverly, Mass.) or Promega Biotech(Madison, Wis.): these reagents were used according to themanufacturer's instruction. For sequencing reactions, a “SEQUENASE DNAII” sequencing kit was used (United States Biochemical Corporation,Cleveland Ohio). Immunoblotting and other reagents were from SigmaChemical Co. (St. Louis, Mo.) or Fisher Scientific (Needham, Mass.).Nitrocellulose filters are obtained from Schleicher and Schuell (Keene,N.H.).

[0375] Synthetic oligonucleotide linkers and primers are prepared usingcommercially available automated oligonucleotide synthesizers (e.g., anABI model 380B-02 DNA synthesizer (Applied Biosystems, Foster City,Calif.)). Alternatively, custom designed synthetic oligonucleotides maybe purchased, for example, from Synthetic Genetics (San Diego, Calif.).cDNA synthesis kit and random priming labeling kits are obtained fromBoehringer-Mannheim Biochemical (BMB, Indianapolis, Ind.).

[0376] Oligonucleotide sequences encoding polypeptides can be eithersynthesized directly by standard methods of oligonucleotide synthesis,or, in the case of large coding sequences, synthesized by a series ofcloning steps involving a tandem array of multiple oligonucleotidefragments corresponding to the coding sequence (Crea; Yoshio et al.;Eaton et al.). Oligonucleotide coding sequences can be expressed bystandard recombinant procedures (Maniatis et al.; Ausubel et al.).

[0377] Alternatively, peptides can be synthesized directly by standardin vitro techniques (Applied Biosystems, Foster City Calif.).

[0378] Common manipulations involved in polyclonal and monoclonalantibody work, including antibody purification from sera, are performedby standard procedures (Harlow, et al.) . Pierce (Rockford, Ill.) is asource of many antibody reagents.

[0379] Recombinant human IFNα (rHuIFNα) and rBoIFNγ was obtained fromGenentech Inc. (South San Francisco, Calif.). The reference preparationof recombinant human IFNα (rHuIFNα) was obtained from the NationalInstitutes of Health: rHuIFNα is commercially available from LeeBiomolecular (San Diego, Calif.).

[0380] All tissue culture media, sera and IFNs used in this study werenegative for endotoxin, as determined by assay with Limulus amebocytelysate (Associates of Cape Cod, Woods Hole, Mass.) at a sensitivitylevel of 0.07 ng/ml.

[0381] A. General ELISA Protocol for Detection of Antibodies.

[0382] Polystyrene 96 well plates Immulon II (PGC) were coated with 5μg/mL (100 μL per well) antigen in 0.1 M carb/bicarbonate buffer, pH9.5. Plates were sealed with parafilm and stored at 4° C. overnight.

[0383] Plates were aspirated and blocked with 300 uL 10% NGS andincubated at 37° C. for 1 hr.

[0384] Plates were washed 5 times with PBS 0.5% “TWEEN-20”.

[0385] Antisera were diluted in 0.1 M PBS, pH 7.2. The desireddilution(s) of antisera (0.1 mL) were added to each well and the plateincubated 1 hours at 37° C. The plates was then washed 5 times with PBS0.5% “TWEEN-20”.

[0386] Horseradish peroxidase (HRP) conjugated goat anti-human antiserum(Cappel) was diluted 1/5,000 in PBS. 0.1 mL of this solution was addedto each well. The plate was incubated 30 min at 37° C., then washed 5times with PBS.

[0387] Sigma ABTS (substrate) was prepared just prior to addition to theplate.

[0388] The reagent consists of 50 mL 0.05 M citric acid, pH 4.2, 0.078mL 30% hydrogen peroxide solution and 15 mg ABTS. 0.1 mL of thesubstrate was added to each well, then incubated for 30 min at roomtemperature. The reaction was stopped with the addition of 0.050 mL 5%SDS (w/v). The relative absorbance is determined at 410 nm.

EXAMPLE 1 Reproductive Functions of IFNτ

[0389] The effect of interferon-τ on the lifespan of the corpus lutemwas examined.

[0390] IFNτ was infused into uterine lumen of ewes at the concentrationsgiven in Table 1. Recombinant human IFNα (rHuIFNα) was infused atsimilar concentrations. In addition, control animals, which receivedcontrol proteins, were also used. The life span of the corpus luteum wasassessed by examination of interestrous intervals, maintenance ofprogesterone secretion, and inhibition of prostaglandin secretion(Davis, et al., 1992). TABLE 1 Effect of Interferons on ReproductivePhysiology Interestrous Interval (days) Interferon Treatment (Means)Control — 17.3 rHuIFNα 100 μg/day 16.0 200 μg/day 16.0 2000 μg/day 19.0OvIFNτ 100 μg/day 27.2

[0391] Comparison of the interestrous intervals for the control animalsand for animals receiving OvIFNτ demonstrate a considerable lengtheningof the interval, when IFNτ is administered at 100 μg/day. On the otherhand, comparison of the interestrous interval for the control animal andfor animals receiving recombinant human IFNα, demonstrated that rHuIFNαhad no meaningful effect.

[0392] These results demonstrate that interferon-τ has the capability ofsignificantly influencing the biochemical events of the reproductivecycle.

EXAMPLE 2 Antiviral Properties of Interferon-τ at Various Stages of theReproductive Cycle

[0393] Conceptus cultures were established using conceptus obtained fromsheep at days 12 through 16 of the estrous cycle. Antiviral activity ofsupernatant from each conceptus culture was assessed using a cytopathiceffect assay (Familetti, et al., 1981). Briefly, dilutions of IFNτ orother IFNs were incubated with Madin-Darby bovine kidney (MDBK) cellsfor 16-18 hours at 37° C. Following incubation, inhibition of viralreplication was determined in a cytopathic effect assay using vesicularstomatitis virus (VSV) as the challenge virus.

[0394] One antiviral unit caused a 50% reduction in destruction of themonolayer, relative to untreated MDBK cells infected with VSV (controlplates). Specific activities were further evaluated using normal ovinefibroblasts (Shnf) in a plaque inhibition assay (Langford, et al.,1981). A minimum of three samples were examined at each time point, andeach sample was assayed in triplicate. The results presented in Table 2are expressed as mean units/ml. TABLE 2 IFNτ Antiviral Activity ofConceptus Cultures and Allantoic and Amniotic Fluids Day SamplesUnits/ml Conceptus Cultures 10 9 <3 12 5 34 13 6 4.5 × 10³ 14 3 7.7 ×10³ 16 12 2.0 × 10⁶ Allantoic Fluid 60 3 1.4 × 10³ 100 4 11 140 3 <3Amniotic Fluid 60 3 22 100 4 <3

[0395] Culture supernatants had increasing antiviral activity associatedwith advancing development of the conceptus (Table 2).

EXAMPLE 3 Expression of IFNτ in Bacteria

[0396] The amino acid coding sequence for OvIFNτ (Imakawa, et al., 1987)was used to generate a corresponding DNA coding sequence with codonusage optimized for expression in E. coli. Linker sequences were addedto the 5′ and 3′ ends to facilitate cloning in bacterial expressionvectors. The nucleotide sequence was designed to include 19 uniquerestriction enzyme sites spaced evenly throughout the coding sequence(FIGS. 1A and 1B).

[0397] The nucleotide sequence was divided into eleven oligonucleotidefragments ranging in sizes of 33 to 75 bases. Each of the elevenoligonucleotides were synthesized on a 380-B 2-column DNA synthesizer(Applied Biosystems) and cloned single- or double-stranded into one ofthe following vectors: “pBLUESCRIPT⁺ (KS)” (Stratagene, LaJolla,Calif.), pTZ18R (Pharmacia, Piscataway, N.J.), or pTZ19R (Pharmacia,Piscataway, N.J.) cloning vectors.

[0398] The vectors were transformed into E. coli K. strain “XL1-BLUE”(recA1 endA1 gyrA96 thi hsdR17 (r_(K) ⁻, m_(K)+) supE44 relA1 λ-(lac),{F′, proAB, laC^(q)ZΔM15, Tn10 (tet^(R)}) which is commerciallyavailable from Stratagene (La Jolla, Calif.). Transformed cells weregrown in L broth supplemented with ampicillin (50 μg/ml).Oligonucleotide cloning and fusion was performed using standardrecombinant DNA techniques.

[0399] Cloning vectors were cut with the appropriate restriction enzymesto insert the synthetic oligonucleotides. The vectors were treated withcalf intestine alkaline phosphatase (CIP) to remove terminal phosphategroups. Oligonucleotides were phosphorylated and cloned, as eithersingle- or double-stranded molecules, into the appropriate vector usingT4 DNA ligase. When single-strands were introduced into cloning vectors,the second strand was completed by the bacterial host followingtransfection.

[0400] For double-stranded cloning, oligonucleotides were first annealedwith their synthetic complementary strand then ligated into the cloningvector. E. coli K12 strains SB221 or NM522 were then transformed withthe ligation. E. coli strain GM119 was used for cloning when themethylation-sensitive StuI and ClaI restriction sites were involved.Restriction analyses were performed on isolated DNA at each stage of thecloning procedure.

[0401] Cloned oligonucleotides were fused into a single polynucleotideusing the restriction digestions and ligations outlined in FIG. 2.Oligonucleotide-containing-DNA fragments were typically isolated afterelectrophoretic size fractionation on low-melting point agarose gels(Maniatis, et al.; Sambrook, et al.; Ausubel, et al.). The resultingIFNτ polynucleotide coding sequence spans position 16 through 531: acoding sequence of 172 amino acids.

[0402] The nucleotide sequence of the final polynucleotide was confirmedby DNA sequencing using the dideoxy chain termination method.

[0403] The full length StuI/SstI fragment (540 bp; FIG. 2) was clonedinto a modified pIN III omp-A expression vector and transformed into acompetent SB221 strain of E. coli. For expression of the IFNτ protein,cells carrying the expression vector were grown in L-broth containingampicillin to an OD (550 nm) of 0.1-1, induced with IPTG for 3 hours andharvested by centrifugation. Soluble recombinant IFNτ was liberated fromthe cells by sonication or osmotic fractionation.

EXAMPLE 4 Expression of IFNτ in Yeast

[0404] The synthetic IFNτ gene, synthesized in Example 3, was flanked atthe 5′ end by an StuI restriction site and at the 3′ end by a SacIrestriction site.

[0405] A. Isolation of the Synthetic IFNτ Gene.

[0406] Two oligonucleotide primers (SEQ ID NO:13 and SEQ ID NO:14) wereused to attach linkers to the synthetic IFNτ gene using polymerase chainreaction. The linker at the 5′ end allowed the placement of thesynthetic IFNτ gene in correct reading with the ubiquitin codingsequence present in the yeast cloning vector pBS24Ub (Chiron Corp.,Emeryville, Calif.). The linker also constructed a ubiquitin-IFNτjunction region that allowed in vivo cleavage of the ubiquitin sequencesfrom the IFNτ sequences. The 5′ oligonucleotide also encoded a SacIIrestriction endonuclease cleavage site. The 3′ oligonucleotide containeda StuI cleavage site.

[0407] The vector carrying the synthetic IFNτ gene (Example 3) wasisolated from E. coli strain “XLI-BLUE” by the alkaline lysis method.Isolated vector was diluted 500-fold in 10 mM Tris, pH 8.0/1 mM EDTA/10mM NaCl. The PCR reaction was performed in a 100 μl volume using Taq DNApolymerase and primers SEQ ID NO:13/SEQ ID NO:14. The amplifiedfragments were digested with StuI and SacII. These digested fragmentswere ligated into the SacII and SmaI sites of “pBLUESCRIPT+(KS).”

[0408] The resulting plasmid was named pBSY-IFNτ. The DNA sequence wasverified using double stranded DNA as the template.

[0409] B. Construction of the Expression Plasmid.

[0410] Plasmid pBSY-IFNτ was digested with SacII and EcoRV and thefragment containing the synthetic IFNτ gene was isolated. The yeastexpression vector pBS24Ub (Sabin, et al.; Ecker, et al.) was digestedwith SalI. Blunt ends were generated using T4 DNA polymerase. The vectorDNA was extracted with phenol and ethanol precipitated (Sambrook, etal., 1989). The recovered linearized plasmid was digested with SacII,purified by agarose gel electrophoresis, and ligated to the SacII-EcoRVfragment isolated from pBSY-IFNτ. The resulting recombinant plasmid wasdesignated pBS24Ub-IFNτ.

[0411] The recombinant plasmid pBS24Ub-IFNτ was transformed into E.coli. Recombinant clones containing the IFNτ insert were isolated andidentified by restriction enzyme analysis. Plasmid DNA from clonescontaining IFNτ coding sequences was used for transformation of S.cerevisiae (Rothstein, 1986). Transformation mixtures were plated onuracil omission medium and incubated for 3-5 days at 30° C. Colonieswere then streaked and maintained on uracil and leucine omission medium(Rothstein, 1986).

[0412] C. Expression Experiments.

[0413] For small-scale expression, a single colony of S. cerevisiaeAB116 containing pBS24Ub-IFNτ was picked from a leucine and uracilomission plate and grown at 30° C. in YEP medium (1% yeast extract, 2%peptone) containing 1% glucose for inducing conditions or 8% glucose fornoninducing conditions. Cell lysates were recovered and subjected toSDS-PAGE in 15% acrylamide, 0.4% bisacrylamide (Sambrook, et al., 1989).The fractionated proteins were visualized by Coomassie blue staining.

[0414] Recombinant IFNτ was visualized specifically by immunoblottingwith monoclonal antibody or polyclonal antiserum against ovine IFNτ uponelectrotransfer of the fractionated cell extract to “NYTRAN” paper(Rothstein, 1986).

[0415] For large-scale expression, pBS24-IFNτ was grown for 24 hours at30° C. in 5× uracil and leucine omission medium containing 8% glucose.This culture was then diluted 20-fold in YEP medium containing 1%glucose and further incubated for another 24-36 hours.

[0416] Cells were harvested by centrifugation, washed in 50 mM Tris, pH7.6,/1 mM EDTA and resuspended in wash buffer containing 1 mM PMSF. Thecells were lysed using a Bead-beater apparatus (Biospec Products,Bartlesville, Okla.). The lysate was spun at 43,000×g for 20 minutes.The supernatant fraction was recovered and subjected to the purificationprotocol described below.

[0417] D. Purification of roIFNτ from Yeast Cell Lysate.

[0418] The supernatant was loaded on a 1×10 cm DEAE column and washedwith 10 mM Tris, pH 8.0. Retained proteins were eluted with a 300 ml, 0to 0.5 M NaCl gradient in 10 mM Tris, pH 8.0. Three-milliliter fractionswere collected. Ten-microliter samples of fractions 17-26 containing therecombinant (roIFNτ) were electrophoretically separated on 15%SDS-polyacrylamide gels. The gels were stained with Coomassie blue.

[0419] Fractions 18, 19, and 20 contained largest amount of roIFNτ.These fractions were loaded individually on a 1.5×90 cm Sephadex S-200column and proteins were resolved in two peaks. Aliquots of each proteinpeak (25 μl) were electrophoretically separated on 15%SDS-polyacrylamide gels and the proteins visualized with Coomassiestaining.

[0420] Purified roIFNτ-containing fractions were combined and the amountof roIFNτ quantified by radioimmunoassay (Vallet, et al., 1988). Totalprotein concentration was determined by using the Lowry protein assay(Lowry, et al., 1951).

[0421] Microsequencing of purified roIFNτ demonstrated identity withnative IFNτ through the first 15 amino acids, confirming that theubiquitin/roIFNτ fusion protein was correctly processed in vivo.

[0422] Purified roIFNτ exhibited 2 to 3×10⁸ units of antiviral activityper milligram of protein (n=3 replicate plates) which is similar to theantiviral activity of IFNτ purified from conceptus-conditioned culturemedium (2×10⁸ U/mg).

EXAMPLE 5 Southern Blot Analysis of Human High Molecular Weight DNA

[0423] Human venous blood samples from healthy donors were collected inheparinized tubes and peripheral blood lymphocytes were isolated bydensity-gradient centrifugation using a Ficoll-Isopaque gradient (1.077g/ml) (Sigma Chemical Co.). High molecular weight (HMW) DNA was isolatedfrom these cells (Sambrook, et al., 1989).

[0424] Two 10 μg samples of HMW DNA were digested with the restrictionendonucleases HindIII or PstI (Promega) for 2 hours at 37° C., and theDNA fragments electrophoretically separated in a 0.8% agarose gel(Bio-Rad, Richmond, Calif.) at 75 volts for 8 hours. The DNA fragmentswere transferred onto a nylon membrane (IBI-InternationalBiotechnologies, Inc., New Haven, Conn.). The membrane was baked at 80°C. for 2 hours and incubated at 42° C. for 4 hours in the followingprehybridization solution: 5×SSC (1×SSC is 0.15 M NaCl and 0.15 M sodiumcitrate), 50% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfatdry milk, 20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 0.5 mg/ml singlestranded herring sperm DNA (Promega).

[0425] The filter was then incubated in a hybridization solution (5×SSC,20% vol/vol formamide, 0.6% (wt/vol) SDS, 0.5% (wt/vol) nonfat dry milk,20 mM Tris-HCl (pH 7.5), 4 mM EDTA, and 2×10⁸ cpm/ml ³²P-labelled OvIFNτcDNA (Imakawa, et al., 1987)) for 18 hours at 42° C. The filter waswashed at 42° C. for 15 minutes with 2×SSC and 0.1% (wt/vol) SDS andexposed to X-ray film (XAR, Eastman Kodak, Rochester, N.Y.) at −80° C.for 48 hours in the presence of an intensifying screen.

[0426] Autoradiography detected a hybridization signal at approximately3.4 kb in DNA digested with PstI and a slightly smaller (≈3.0 kb)fragment in the HindIII digested DNA. These results indicate thepresence of human DNA sequences complementary to the OvIFNτ cDNA probe.

EXAMPLE 6 Isolation of Partial Sequence of Human IFNτ cDNA by PCR

[0427] Two synthetic oligonucleotides (each 25-mer), corresponding tothe nucleotides in the DNA sequence from 231 to 255 (contained in SEQ IDNO:13) and 566 to 590 (contained in SEQ ID NO:14) of OvIFNτ cDNA(numbering relative to the cap site, Imakawa, et al., 1987) weresynthesized. These primers contained, respectively, cleavage sites forthe restriction endonucleases PstI and EcoRI. SEQ ID NO:13 was modifiedto contain the EcoRI site, which begins at position 569.

[0428] DNA was isolated from approximately 1×10⁵ plaque forming units(pfu) of the following two cDNA libraries: human term placenta(Clontech, Inc., Palo Alto, Calif.) and human term cytotrophoblast (Dr.J. F. Strauss, University of Pennsylvania, Philadelphia Pa.). The DNAwas employed in polymerase chain reaction (PCR) amplifications (Mullis;Mullis, et al.; Perkin Elmer Cetus Corp. Norwalk Conn.). Amplificationreactions were carried out for 30 cycles (45° C., 1 m; 72° C., 2 m; 94°C., 1 m) (thermal cycler and reagents, Perkin Elmer Cetus) using primersSEQ ID NO:13/SEQ ID NO:14.

[0429] Amplification products were electrophoretically separated (100volts in a 1.5% agarose gel (Bio-Rad)) and transferred onto a nylonmembrane (IBI). The membrane was baked at 80° C. for 2 hours andprehybridized and hybridized with ³²P-labelled OvIFNτ cDNA as describedabove. The membrane was washed in 5×SSC/0.1% (wt/vol) SDS for 5 minutesat 42° C. and in 2×SSC/0.1% (wt/vol) SDS for 2 minutes at 42° C. It wasthen exposed at −80° C. to “XAR” (Eastman Kodak) X-ray film for 24 hoursin the presence of an intensifying screen. An amplification product thathybridized with the labelled probe DNA was detected.

[0430] PCR was performed again as directed above. Amplified productswere digested with the restriction endonucleases EcoRI and PstI(Promega) for 90 minutes at 37° C. The resulting DNA fragments wereelectrophoretically separated as described above and the band containingthe IFNτ amplification product was excised from the gel. DNA fragmentswere recovered by electroelution, subcloned into EcoRI/PstIdigested-dephosphorylated plasmid pUC19 and transformed into E. colistrain JM101 (Promega) by calcium chloride method (Sambrook, et al.,1989). The plasmids were isolated and the inserted amplification productsequenced using the dideoxy termination method (Sanger, et al., 1977;“SEQUENASE” reactions, United States Biochemical, Cleveland, Ohio).Nucleotide sequences were determined, and comparison of these as well asthe deduced amino acid sequences to other IFN sequences were performedusing “DNA STAR SOFTWARE” (Madison, Wis.).

[0431] Comparison of the sequences of these clones revealed thefollowing four different clones: from the human placental library,HuIFNτ6 (299 bp), HuIFNτ7 (288 bp) and HuIFNτ4 (307 bp), which exhibit95% identity in their nucleotide sequences; from the cytotrophoblastlibrary clond CTB 35 (HuIFNτ5; 294 basepairs), which shares 95% and 98%identity with HuIFNτ6 and HuIFNτ4, respectively.

EXAMPLE 7 Isolation of Full-Length Human IFNτ Genes

[0432] Ten micrograms PBMC HMW DNA was digested with restrictionendonuclease EcoRI and subjected to electrophoretic analysis in a 0.8%agarose gel. A series of samples containing ranges of DNA fragmentssized 1.5 to 10 kb (e.g., 1.5 to 2.5 kb, 2.5 kb to 3 kb) were excisedfrom the gel. The DNAs were electroeluted and purified. Each DNA samplewas amplified as described above using the OvIFNτ primers. The DNAmolecules of any sample that yielded a positive PCR signal were clonedinto gtll (the subgenomic λgt11 library).

[0433] A. PCR Identification of Clones Containing SequencesComplementary to OvIFNτ.

[0434] The λgt11 phage were then plated for plaques and plaque-lifthybridization performed using the ³²P-labelled OvIFNτ cDNA probe.Approximately 20 clones were identified that hybridized to the probe.

[0435] Plaques that hybridized to the probe were further analyzed by PCRusing the OvIFNτ primers described above. Six plaques which generatedpositive PCR signals were purified. The phage DNA from these clones wasisolated and digested with EcoRI restriction endonuclease. The DNAinserts were subcloned into pUC19 vectors and their nucleotide sequencesdetermined by dideoxy nucleotide sequencings.

[0436] B. Hyhridization Identification of Clones Containing SequencesComplementary to PCR-Positive Phage.

[0437] Recombinant phage from the λgt11 subgenomic library werepropagated in E. coli Y1080 and plated with E. coli Y1090 at a densityof about 20,000 plaques/150 mm plate. The plates were overlaid withduplicate nitrocellulose filters, which were hybridized with a³²P-labelled probe from one of the six human IFNτ cDNA clones isolatedabove.

[0438] Clones giving positive hybridization signals were furtherscreened and purified. The phage DNAs from hybridization-positive cloneswere isolated, digested with EcoRI, subcloned into pUC19 vector andsequenced. The sequence information was then analyzed.

[0439] 1. HuIFNτ1

[0440] Three clones yielded over-lapping sequence information for over800 bases relative to the mRNA cap site (clones were sequenced in bothorientations). The combined nucleic acid sequence information ispresented as SEQ ID NO:11 and the predicted protein coding sequence ispresented as SEQ ID NO:12. Comparison of the predicted mature proteinsequence (SEQ ID NO:12) of this gene to the predicted protein sequenceof OvIFNτ is shown in FIG. 3.

[0441] 2. HuIFNτ2, HuIFNτ3

[0442] Two additional clones giving positive hybridization signals(HuIFNτ2 and HuIFNτ3) were also screened, purified, and phage DNAssubcloned and sequenced as above. The sequences of these two clones arepresented in FIGS. 19A and 19B. As can be appreciated in FIGS. 19A and19B, the nucleotide sequence of both clones (HuIFNτ2 and HuIFNτ3) ishomologous to that of HuIFNτ1 and OvIFNτ.

[0443] HuIFNτ2 (SEQ ID NO:29), may be a pseudo-gene, as it appears tocontain a stop codon at position 115-117. The sequence, SEQ ID NO:29, ispresented without the leader sequence. The leader sequence is shown inFIG. 20A. As can be seen from the HuIFNτ2 sequence presented in FIG.20A, the first amino acid present in mature HuIFNτ1 (a CYS residue) isnot present in the HuIFNτ2 sequence. Accordingly, the predicted aminoacid sequence presented as SEQ ID NO:29 corresponds to a mature IFNτprotein with the exceptions of the first CYS residue and the internalstop codon.

[0444] The internal stop codon in the nucleic acid coding sequence canbe modified by standard methods to replace the stop codon with an aminoacid codon, for example, encoding GLN. The amino acid GLN is present atthis position in the other isolates of human IFNτ (HuIFNτ). Standardrecombinant manipulations also allow introduction of the initial CYSresidue if so desired.

[0445] HuIFNτ3 (SEQ ID NO:31), appears to encode a human IFNτ protein.The translated amino acid sequence of the entire protein, including theleader sequence, is presented as SEQ ID NO:32. The translated amino acidsequence of the mature protein is presented as SEQ ID NO:34.

EXAMPLE 8 Analysis of the Presence of HuIFNτ mRNA by RT-PCR

[0446] Human placental cDNA libraries and an ovine cDNA library,constructed from day 15-16 conceptuses, were analyzed by hybridizationto the OvIFNτ cDNA probe, described above. cDNAs were size-fractionatedon agarose gels and transferred to filters (Maniatis, et al.; Sambrook,et al.). Southern blot analysis with OvIFNτ probe showed that theautoradiographic signals from human cDNA libraries were approximately1/100 of the signal obtained using the OvIFNτ cDNA library.

[0447] The presence of HuIFNτ mRNA in human term placenta and amniocytes(26 weeks, 2 million cells) was analyzed by using reversetranscriptase-PCR (RT-PCR) method (Clontech Laboratories, Palo AltoCalif.).

[0448] Total cellular RNA (tcRNA) isolated from human placenta,amniocytes and ovine conceptuses were reverse transcribed using theprimer SEQ ID NO:14. The primer SEQ ID NO:13 was then added to thereaction and polymerase chain reaction carried out for 40 cycles. ThePCR products were size fractionated on agarose gels and transferred tofilters. The DNA on the filters was hybridized with ³²P-labelled OvIFNτand HuIFNτ cDNAs. The results of these analyses demonstrate the presenceof human IFNτ mRNA in the feto-placental annex. The aminocytes alsoexpressed the messages corresponding to OvIFNτ primers and human probe.

[0449] In addition, a RT-PCR analysis for the presence of HuIFNτ wasapplied to the tcRNA isolated from human adult lymphocytes. Adensitometric analysis revealed that IFNτ mRNA exists in lymphocytes.

EXAMPLE 9 In Situ Hybridization

[0450] A. Tissue

[0451] Slides of semiserial 5-μ paraffin embedded sections from fourhealthy, different term and first trimester human placentas wereexamined.

[0452] B. cRNA Probe Preparation

[0453] From the cDNA clone isolated from OvIFNτ amplified library afragment corresponding to the OvIFNτ cDNA bases #77-736 (base #1 is capsite; open reading frame of OvIFNτ cDNA is base #81-665; FIG. 7) wassubcloned into the transcription vector, pBS (New England Biolabs).Several pBS clones were isolated, subcloned, and their nucleotidessequenced. From this clone a 3′ fragment (bases #425-736) was excisedusing the restriction endonucleases NlaIV and EcoRI and subcloned intothe transcription vector pBS. This vector was designated pBS/OvIFNτ.

[0454] After linearization of the pBS/OvIFNτ plasmid, an antisense cRNAprobe was synthesized by in vitro transcription (Sambrook, et al., 1989)using T₇ RNA polymerase (Stratagene). A trace amount of ³H-CTP(NEN-DuPont, Cambridge, Mass.) was used in the transcription reaction.dUTP labeled with digoxigenin (Boehringer-Mannheim, Indianapolis, Ind.)was incorporated into the cRNA and yield was estimated through TCAprecipitation and scintillation counting.

[0455] C. Hybridizatiorn

[0456] In situ hybridization was performed using the anti-sense RNAprobe, as described by Lawrence, et al. (1985) with the followingmodifications. Deparaffinized and hydrated sections were prehybridizedfor 10 minutes at room temperature in phosphate buffered saline (PBS)containing 5 mM MgCl₂. Nucleic acids in the sections were denatured for10 minutes at 65° C. in 50% formamide/2×SSC. Sections were incubatedovernight at 37° C. with a hybridization cocktail (30 μl/slide)containing 0.3 μg/ml digoxigenin-labelled cRNA probe and then washed for30 minutes each at 37° C. in 50 formamide/1×SSC. Final washes wereperformed for 30 minutes each at room temperature in 1×SSC and 0.1×SSC.The sections were blocked for 30 minutes with 0.5% Triton X-100 (Sigma)and 0.5% non-fat dry milk.

[0457] Hybridization signal was detected using purified sheepantidioxigenin Fab fragments conjugated to alkaline phosphatase(Boehringer-Mannheim). After unbound antibody was removed, nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate substrate (Promega) andlevamisole (Bector Laboratories, Burlingame, Calif.) were added forsignal detection via colorimetric substrate generation. The tissues werecounterstained in methyl green (Sigma), dehydrated, and mounted.

[0458] As a control, some tissue sections were pretreated with 100 μg/mlof pancreatic RNaseA (Sigma) for 30 minutes at 37° C. The RNase wasinactivated on the slide with 400 units of RNase inhibitor (Promega).The slides were then washed twice in 250 ml of PBS/5 mM MgCl₂. In othercontrol experiments, tRNA (Sigma) was substituted for the digoxigeninprobes.

[0459] Specific hybridization was observed in all term and firsttrimester placental tissues in three separate experiments with variousOvIFNτ cRNA probe concentrations and blocking reagents.

[0460] First trimester placental villi composed of an outer layer ofsyncytiotrophoblast, an underlying layer of cytotrophoblast, and acentral stromal region with various types of mesenchymal cells,displayed the highest transcript level of IFNτ in the cytotrophoblastcells. Less intense but detectable levels were present in both thesyncytiotrophoblast and stromal cells. A similar pattern of transcriptexpression was demonstrated in the placental villi of term tissue butthe level of signal detection was low. First trimester extravilloustrophoblast displayed the highest amount of message and stained positivewhen present in the maternal blood spaces.

EXAMPLE 10 Antiviral Activity of IFNτ

[0461] The relative specific activity of OvIFNτ, purified tohomogeneity, was evaluated in antiviral assays. The antiviral assayswere performed essentially as described above in Example 2. Specificactivities are expressed in antiviral units/mg protein obtained fromantiviral assays using either Madin-Darby bovine kidney (MDBK) cells orsheep normal fibroblasts (Shnf). All samples were assayed simultaneouslyto eliminate interassay variability. The results, presented in Table 3,are the means of four determinations where the standard deviation wasless than 10% of the mean. TABLE 3 Antiviral Activity of IFNτ and KnownIFNs Specific Activies MDBK Shnf OvIFNτ   2 × 10⁸ 3 × 10⁸ rBoIFNα   6 ×10⁷ 1 × 10⁷ rBoIFNΥ 4.5 × 10⁶ 3 × 10⁶ NIH rHuIFNα 2.2 × 10⁸ 2.2 × 10⁸  rHuIFNα 2.9 × 10⁵ 4.3 × 10⁵  

[0462] IFNτ had a higher specific activity than either rBoIFNα orrBoIFNγ (Table 3). The NIH standard preparation of rHuIFNα had a similarspecific activity, while a commercial preparation of rHuIFNα exhibitedlow specific antiviral activity. Comparable relative antiviral activitywas demonstrated using either bovine or ovine cells.

EXAMPLE 11 Anti-Retroviral Activity and Cytotoxic Effects of IFNτ

[0463] Highly purified OvIFNτ was tested for anti-retroviral andcytotoxic effects on feline peripheral blood lymphocytes exposed to thefeline immunodeficiency retrovirus. This lentivirus produces a chronicAIDS-like syndrome in cats and is a model for human AIDS (Pederson, etal., 1987). Replication of the virus in peripheral blood lymphocytes ismonitored by reverse transcriptase activity in culture supernatants overtime. The data from these assays are presented in Table 4. TABLE 4Effect of OvIFNτ on FIV Replication IFNτ Concentration (ng/ml) RTActivity (cpm/ml) Harvest Days Experiment 1 Day 2 Day 5 Day 8 Day 12 Day15 0.00 93,908 363,042 289,874 171,185 125,400 0.62 77,243 179,842172,100 218,281 73,039 1.25 94,587 101,873 122,216 71,916 50,038 2.5063,676 72,320 140,783 75,001 36,105 5.00 69,348 82,928 90,737 49,54636,299 Harvest Days Experiment 2 Day 2 Day 5 Day 8 Day 13 Day 17 0.0210,569 305,048 279,556 500,634 611,542 2.5 121,082 106,815 108,882201,676 195,356 5.0 223,975 185,579 108,114 175,196 173,881 10.0 167,425 113,631 125,131 131,649 129,364 20.0  204,879 80,399 59,45878,277 72,179 40.0  133,768 54,905 31,606 72,580 53,493

[0464] Addition of OvIFNτ produced a rapid, dose-dependent decrease inreverse transcriptase (RT) activity (Table 4). While concentrations aslow as 0.62 ng/ml of IFNτ inhibited viral replication, much higherconcentrations (40 ng/ml) having greater effects on RT-activity werewithout toxic effects on the cells. The results suggest that replicationof the feline immunodeficiency virus was reduced significantly comparedto control values when cells were cultured in the presence of OvIFNτ.

[0465] IFNτ appeared to exert no cytotoxic effect on the cells hostingthe retrovirus. This was true even when IFNτ was present at 40 ng per mlof culture medium.

EXAMPLE 12 Effects of IFNτ on HIV Infected Human Peripheral Lympohocytes

[0466] IFNτ was also tested for activity against HIV infection in humancells. Human peripheral blood lymphocytes, which had been infected withHIV (Crowe, et al.), were treated with varying concentrations of OvIFNτ.Replication of HIV in peripheral blood lymphocytes was monitored byreverse transcriptase activity in culture supernatants over time.Reverse transcriptase activity was measured essentially by the method ofHoffman, et al. The data from these assays are presented in Table 5.TABLE 5 Effect of OvIFNτ on HIV Replication in Human PeripheralLymphocytes IFNτ RT Activity Concentration Day 6 Day 10 (ng/ml) cpm/ml %Reduction cpm/ml % Reduction 0 4,214 — 25,994 — 10 2,046 51 9,883 62 501,794 57 4,962 81 100 1,770 58 3,012 88 500 1,686 60 2,670 90 1000 1,49964 2,971 89

[0467] As shown in Table 5, concentrations of OvIFNτ producedsignificant antiviral effects. A concentration of only 10 ng/ml resultedin over a 50% reduction in RT activity after only six days. Aconcentration of 500 ng/ml resulted in a 90% reduction in RT activitywithin 10 days.

[0468] The viability of human peripheral blood lymphocytes aftertreatment with IFNτ, over a range of concentrations for 3-13 days, wasevaluated by trypan blue exclusion. The results of this viabilityanalysis are presented in Table 6. TABLE 6 Effect of OvIFNτ on Viabilityof HIV Infected Human Peripheral Lymphocytes IFNτ Concentration ViableCells/ml × 10⁵ (ng/ml) Day 3 Day 6 Day 13 0 16.0 7.5 5.3 10 13.0 7.5 6.050 13.0 11.5 9.0 100 15.0 8.5 9.5 500 16.5 12.0 11.0 1000 21.9 9.5 8.5

[0469] The data presented in Table 6 show no evidence of cytotoxiceffects attributable to the administration of IFNτ.

EXAMPLE 13 Inhibition of Cellular Growth

[0470] The effects of IFNτ on cellular growth were also examined.Anti-cellular growth activity was examined using a colony inhibitionassay. Human amnion (WISH) or MDBK cells were plated at low celldensities to form colonies originating from single cells. Cells werecultured at 200 or 400 cells/well in 24 well plates in HMEM supplementedwith 2% fetal bovine serum (FBS) and essential and nonessential aminoacids. Various dilutions of interferons were added to triplicate wells,and the plates were incubated for 8 days to allow colony formation.Colonies were visualized after staining with crystal violet, andcounted. Cell cycle analysis was performed with HMEM containing 0.5%“spent” media for an additional 7 days. WISH cells were used withoutbeing synchronized.

[0471] For examination of IFNτ activity, cells were replated at 2.5×10⁵cells/well in HMEM with 10% FBS in 6 well plates. Various dilutions ofOvIFNτ alone or in combination with peptides were added to achieve afinal volume of 1 ml. Plates were incubated at 37° C. in 5% Co₂ for 12,15, 18, 24, or 48 hours. Cells were treated with trypsin, collected bylow speed centrifugation and washed. The cell pellet was blotted dry and250 μl of nuclear staining solution (5 mg propidium iodide, 0.3 ml NP40and 0.1 gm sodium citrate in 100 ml distilled H₂O) was added to eachtube. The tubes were incubated at room temperature. After 10 minutes,250 μl of RNase (500 units/ml in 1.12% sodium citrate) was added pertube and incubated an additional 20 minutes. Nuclei were filteredthrough 44 μm mesh, and analyzed on a FACStar (Becton Dickinson,Mountain View, Calif.) using the DNA Star 2.0 software.

[0472] In the cellular growth assay using colony formation of both thebovine epithelial line, MDBK, and the human amniotic line, WISH, OvIFNτinhibited both colony size and number. Ovine IFNτ was more effectivethan human IFNα on the human cell line; thus, it is very potent incross-species activity. Its activity was dose-dependent, and inhibitionof proliferation could be observed at concentrations as low as 1unit/ml. Concentrations as high as 50,000 units/ml (units of antiviralactivity/ml) stopped proliferation, while cell viability was notimpaired.

[0473] Cell cycle analysis by flow cytometry with propidiumiodide-stained WISH cells revealed an increased proportion of cells inG2/M after 48 hours of OvIFNτ treatment. IFNτ, therefore, appears toinhibit progress of cells through S phase. Ovine IFNτ antiproliferativeeffects can be observed as early as 12 hours after the initiation ofculture and are maintained through 6 days.

[0474] The results presented above demonstrate both theantiproliferative effect of IFNτ as well as its low cytotoxicity.

EXAMPLE 14 Further Antiproliferative Effects of IFNτ

[0475] The antiproliferative effects of OvIFNτ were studied for a ratcell line and a bovine cell line. The rate of ³H-thymidine incorporationwas used to assess the rate of cellular proliferation.

[0476] Rat (MtBr7 .c5) or bovine kidney (MDBK) cells were seeded inphenol red-free DME-F12 medium supplemented with 3% dextran-coatedcharcoal stripped Controlled Process Serum Replacement 2 (CPSR 2, Sigma)and 5% dextran-coated charcoal stripped fetal bovine serum (FBS). Afterattaching for approximately 15-18 hours, the cells were washed once withserum-free DME-F12 medium. The medium was replaced with phenol red-freeDME-F12 medium supplemented with 3% stripped CPSR2, 1% stripped FBS(“3/1” medium) or 3/1 medium containing OvIFNτ at various units ofantiviral activity as determined in the vesicular stomatitis viruschallenge assay for interferons (Example 2). Media containing a similardilution of buffer (undiluted buffer=10 mM Tris, 330 mM NaCl, [TS]), inwhich the OvIFNτ was dissolved was used for controls.

[0477] Cells were pulse labeled with ³H-thymidine for 2 hours atapproximately 48 hours post-treatment. The trichloroacetic acid (TCA)precipitable incorporated counts were determined by scintillationcounting. Three replicates were included per treatment. Mean values forOvIFNτ treatments were compared to samples containing comparabledilutions of carrier TS buffer. Results of these experiments are shownin Table 7. TABLE 7 ³H-Thymidine Incorporation % Reduction ³H-ThymidineTreatment Incorporation Experiment 1: MtBr7 .c5 (Rat) 3/1 — 10³ uOvIFNτ/ml 0 (+12) 1:5000 TS — 10⁴ u OvIFNτ/ml 24 1:500 TS — 10⁵ uOvIFNτ/ml 87 Experiment 2: MDBK 3/1 — 10³ u OvIFNτ/ml 74 1:5000 TS — 10⁴u OvIFNτ/ml 83 1:500 TS — 10⁵ u OvIFNτ/ml 83

[0478] As can be seen from Table 7, OvIFNτ drastically reduced the rateof cellular proliferation (based on thymidine incorporation) for each ofthe cell lines tested.

EXAMPLE 15 Antiproliferative Effects of IFNτ on Human Tumor Cell Lines

[0479] The antiproliferative activity of OvIFNτ on human tumor celllines was evaluated by measuring the rate of ³H-thymidine incorporationinto cells which have been treated with OvIFNτ.

[0480] For experiments on tumor lines that grow in suspension, 1 ml ofcells were plated at from 2.5−5×10⁵ cells/well in 24-well plates.Triplicate wells received either the appropriate media, 100, 1,000 or10,000 units/ml of OvIFNτ or equivalent antiviral concentrations ofrHuIFNα2A (Lee Biomolecular). After 48 hours of incubation, cells werecounted and viability assessed by trypan blue exclusion.

[0481] Adherent tumor lines were plated at 2.5×10⁵ cells/well in 1 ml in6-well plates. They received interferon treatments as just described,but were trypsinized prior to counting.

[0482] Significant differences between treatments were assessed by ananalysis of variance followed by Scheffe's F-test. Cell cycle analysiswas performed by flow cytometry using propidium iodide.

[0483] A. Breast Adenocarcinoma Cells.

[0484] Human MCF7 breast adenocarcinoma cells were seeded fromlogarithmically growing cultures in phenol red-free DME-F12 mediumsupplemented with 3% dextran-coated charcoal stripped CPSR and 5%dextran-coated FBS. After attaching for approximately 15-18 hours, thecells were washed once with serum-free DME-F12 medium. The medium wasreplaced with phenol red-free DME-F12 medium supplemented with 3%stripped CPSR2, 1% stripped FBS (“3/1” medium) or 3/1 medium containingOvIFNτ at the indicated number of units of antiviral activity asdetermined in the vesicular stomatitis virus challenge assay forinterferons. Media containing a similar dilution of buffer (undilutedbuffer=10 mM Tris, 330 mM NaCl [TS]) was used for controls. Cells werepulse labeled with ³H-thymidine for 2 hours at approximately 48 hourspost-treatment.

[0485] The trichloroacetic acid (TCA) precipitable incorporated countswere determined by scintillation counting. Three replicates wereincluded per treatment. Mean values for OvIFNτ treatments were comparedto samples containing comparable dilutions of carrier TS buffer. Theresults of these analyses are shown in Table 8. TABLE 8 ³H-ThymidineIncorporation % Reduction ³H-Thymidine Treatment Incorporation MCF7Human 3/1 — 10³ u OvIFNτ/ml 35 1:5000 TS — 10⁴ u OvIFNτ/ml 53 1:500 TS —10⁵ u OvIFNτ/ml 70

[0486] As can be seen from the results shown in Table 8, OvIFNτ was ableto substantially reduce the rate of ³H-thymidine incorporation in thehuman carcinoma cell line. This demonstrates the efficacy of OvIFNτ ininhibiting tumor cell proliferation, in particular, mammary tumor cellproliferation.

[0487] B. Human Promyelocytic Leukemia.

[0488] A comparison of the antiproliferative effects of OvIFNτ and IFNαwas conducted using HL-60 (human leukemia) cells (Foa, et al.; Todd, etal.) essentially as described above for MDBK cells. Both OvIFNτ andrHuIFNα inhibit HL-60 cell proliferation. Results of one of threereplicate experiments are presented as mean % growth reduction±SD inFIG. 4. FIG. 4 shows that both OvIFNτ and IFNα were able to drasticallyreduce growth of HL-60 cells. The growth reduction for each compoundexceeded 60% for each concentration tested. At 10,000 units/ml, OvIFNτcaused an approximately 80% reduction in growth while IFNα caused a 100%reduction in growth.

[0489] However, the data presented in FIG. 4 reveal, that a substantialfactor in the ability of IFNα to reduce growth was its toxic effect onthe cells. At 10,000 units/ml, the toxicity of IFNα resulted in lessthan 25% of the cells remaining viable. By contrast, nearly 100% of thecells remained viable when OvIFNτ was applied at 10,000 units/ml.

[0490]FIG. 5 presents data demonstrating that rHuIFNα is cytotoxic. Inthe figure, results of one of three replicate experiments are presentedas mean % viability±SD.

[0491] C. Human Cutaneous T Cell Lymphoma.

[0492] The cutaneous T cell lymphoma, HUT 78, responded similarly toHL-60 when treated with IFNτ (FIG. 9). Both OvIFNτ and rHuIFNo reduceHUT 78 cell growth, but 10,000 units/ml of rHuIFNα decreased the cellnumber below that originally plated (5×10⁵). This is indicative of areduction in cell viability to approximately 60%.

[0493] Cell cycle analysis (performed by cell flow cytometry) revealedan increased proportion of cells in G2/M phase of the cell cycle upon 48hours of treatment with both interferons (Table 9). In Table 9 theresults from one of three replicate experiments are presented as thepercentage of cells in each phase of the cell cycle. 10,000 events wereanalyzed per sample.

[0494] This result is likely due to the slower progress of cells throughthe cell cycle. In the sample treated with 10,000 units/ml of rHuIFNα, alarge percentage of events with low forward and high side scatter,identifying dead cells, were present. This is consistent with the dataobtained from proliferation experiments, where only OvIFNτ inhibited HUT78 proliferation without toxicity. TABLE 9 HUT 78 Cell Cycle AnalysisTreatment (units/ml) G0/G1 S G2/M Media 44.43 49.95 5.61 100 OvIFNτ44.35 47.45 8.20 100 rHuIFNα 40.01 57.53 2.45 1,000 OvIFNτ 41.29 50.508.21 1,000 rHuIFNα 41.73 44.91 13.36 10,000 OvIFNτ 42.79 42.61 14.6010,000 rHuIFNα 18.01 71.31 10.67 (cell death)

[0495] D. Human T Cell Lymphoma.

[0496] The T cell lymphoma cell line H9 was slightly less sensitive tothe antiproliferative effects of the IFNs than the tumor cell linesdescribed above. Results of one of three replicate experiments arepresented in FIG. 10 as mean % growth reduction±SD. While rHuIFNα wasnot toxic to the H9 cells, it failed to inhibit cell divisionsignificantly at any of the concentrations examined. In contrast, OvIFNτwas observed to reduce H9 growth by approximately 60% (FIG. 10). Thus,only OvIFNτ is an effective growth inhibitor of this T cell lymphoma.

[0497] The results presented above demonstrate both theantiproliferative effect of IFNτ as well as its low cytotoxicity.

EXAMPLE 16 Preliminary In Vivo Treatment with OvIFNτ

[0498] Three groups of 4 C57Bl/6 mice per group were given 2.5×10⁴B16-F10 cells via the tail vein: B16-F10 is a syngeneic mousetransplantable tumor selected because of its high incidence of pulmonarymetastases (Poste, et al., 1981). Interferon treatment was initiated 3days after the introduction of the tumor cells. Each mouse received 100μl of either PBS alone, PBS containing 1×10⁵ units of OvIFNτ, or PBScontaining 1×10⁵ units of recombinant murine IFNα (MuIFNα), i.v. per dayfor 3 consecutive days.

[0499] Mice were sacrificed at 21 days and the lungs were preserved in10% buffered formalin. The frequency of pulmonary metastases werecompared between control mice (PBS), OvIFNτ-treated mice, andMuIFNα-treated mice. The results of these in vivo administrationsdemonstrated that OvIFNτ dramatically reduced B16-F10 pulmonary tumors.These results support the use of IFNτ as an efficacious antineoplasticagent in vivo.

EXAMPLE 17 Competitive Binding of IFNτ Peptide Fragments

[0500] A. The Ability of IFNτ-Based Peptides to Block IFNτ and IFN-αAntiviral Activity.

[0501] Overlapping synthetic peptides were synthesized corresponding tothe entire IFNτ sequence (FIG. 6). Average hydropathicity values werecalculated by taking the sum of the hydropathy values for each aminoacid divided by the total number of amino acids in each sequence.Hydropathy values were taken from Kyte, et al. (1982).

[0502] These peptides were of approximately the same molecular weightbut differed slightly in overall hydrophilicity. Despite thisdifference, all peptides were antigenic as demonstrated by theproduction of rabbit antisera with titers greater than 1:3,000 asassessed by ELISA (Harlow, et al.).

[0503] The peptides were used to inhibit the antiviral activity (Example2) of OvIFNτ and rBoIFNα. The results of this analysis are presented inFIG. 12: 1 mM N- and C-terminal peptides both effectively blocked theantiviral activity of OvIFNτ using MDBK cells. A third peptide,representing amino acids 62-92, also reduced IFNτ antiviral activity(70% inhibition). The peptide OvIFNτ (119-150) showed minimal inhibitoryactivity. The OvIFNτ (34-64) and (90-122) peptides had no apparentinhibitory activity.

[0504] Peptide inhibition of OvIFNτ antiviral activity was also examinedas follows. Monolayers of Madin Darby bovine kidney cells were incubatedwith 40 units/ml OvIFNτ in the presence or absence of variousconcentrations of OvIFNτ peptides (see FIG. 13). Results in FIG. 13 areexpressed as the percent of control antiviral activity: that is, in theabsence of any competing peptide. Data presented are the means of 6replicate experiments. The data demonstrate that inhibition by OvIFNτ(1-37), (62-92), (119-150), and (139-172) were significantly differentthan OvIFNτ (34-64) and (90-122) at 10⁻³ M and 3×10⁻³ M. OvIFNτ(139-172) was significantly different than all other peptides at 10⁻³ M.Significance was assessed by analysis of variance followed by Scheffe'sF test at p<0.05. Thus, OvIFNτ (1-37) (62-92), (119-150), and (139-172),in particular (139-172), may represent receptor binding regions forIFNτ.

[0505] The ability of the OvIFNτpeptides to inhibit bovine IFNα (BoIFNα)antiviral activity was examined as follows. Monolayers of Madin Darbybovine kidney cells were incubated with 40 units/ml bovine IFNα in thepresence or absence of various concentrations of OvIFNτ peptides. Theresults are presented in FIG. 14 and are expressed as the percent ofcontrol antiviral activity in the absence of OvIFNτ peptides. The datapresented are the means of 4 replicate experiments. The results indicatethat inhibition by OvIFNτ (62-92), (119-150), and (139-172), weresignificantly different from OvIFNτ (1-37), (34-64) and (90-122) at 10⁻³M. OvIFNτ (139-172) was significantly different than OvIFNτ (1-37),(34-64) and (90-122) at 3×10⁻³ M. Significance was assessed by analysisof variance followed by Scheffe's F test at p<0.05. Thus, OvIFNτ(62-92), (119-150), and (139-172), in particular (139-172), mayrepresent common receptor binding regions for IFNτ and bovine IFNα.

[0506] Peptide inhibition by OvIFNτ peptides of human IFNα antiviralactivity was also examined. Monolayers of Madin Darby bovine kidneycells were incubated with 40 units/ml human IFNα in the presence orabsence of various concentrations of OvIFNτ peptides. The results areexpressed as the percent of control antiviral activity in the absence ofOvIFNτ peptides. The data are presented in FIG. 15 and are the means of3 replicate experiments. OvIFNτ (139-172) was significantly differentfrom all other peptides at 10⁻³ M. Significance was assessed by analysisof variance followed by Scheffe's F test at p<0.05. Thus, OvIFNτ(139-172) may represent a common receptor binding region for IFNτ andvarious IFNα(s).

[0507] The OvIFNτ peptides described above appear to have no effect onthe antiviral activity of IFNγ. Peptide inhibition of bovine IFNγantiviral activity was evaluated as follows. Monolayers of Madin Darbybovine kidney cells were incubated with 40 units/ml bovine IFN gamma inthe presence or absence of various concentrations of OvIFNτ peptides.Results are expressed as the percent of control antiviral activity inthe absence of OvIFNτ peptides. The data are presented in FIG. 16 andare the means of 3 replicate experiments. There were no significantdifferences among peptides as assessed by analysis of variance followedby Scheffe's F test at p<0.05.

[0508] The two synthetic peptides OvIFNτ (1-37) and OvIFNτ (139-172)also blocked OvIFNτ anti-FIV and anti-HIV activity. Reversetranscriptase (RT) activity (Examples 12 and 13) was monitored over a 14day period in FIV-infected FET-1 cells (1×10⁶/ml) and HIV-infected HPBL(1×10⁶/ml). Control cultures received no OvIFNτ. OvIFNτ was used at 100ng/ml, and peptides were used at 200 μM. Data from a representativeexperiment are expressed as cpm/ml culture supernatant and are presentedfor FIV infected cells, FIG. 11A, and HIV infected cells, FIG. 11B. Boththe N- and C-terminus of OvIFNτ appear to be involved in itsanti-retroviral activity. While both peptides blocked FIV RT activity,only the C-terminal peptide, OvIFNτ (139-172), was an efficientinhibitor of vesicular stomatitis virus activity on the feline cellline, Fc9. Thus the C-terminal regions of type I IFNs may bind to commonsite on the type I IFN receptor, while the N-terminal region may beinvolved in the elicitation of unique functions.

[0509] B. Anti-Peptide Sera

[0510] The ability of anti-peptide antisera to inhibit OvIFNτ antiviralactivity was also determined. Antipeptide antisera inhibition of OvIFNτantiviral activity was evaluated as follows. Monolayers of MDBK cellswere incubated with 20 units/ml of OvIFNτ in the presence a 1:30dilution of either preimmune sera or antisera to each of the OvIFNτpeptides described above. In FIG. 17 the data from duplicate experimentsare presented as the mean percent inhibition of OvIFNτ antiviralactivity produced by antipeptide antisera relative to the appropriatepreimmune sera±standard error. Significant differences were assessed byanalysis of variance followed by Scheffe's F test at p<0.05. Consistentwith peptide inhibition of antiviral activities, sera containingantibodies immunoreactive to OvIFNτ (1-37), OvIFNτ (62-92), and OvIFNτ(139-172) were also the most effective inhibitors of OvIFNτ antiviralactivity, with antibodies directed against the N-terminal and C-terminalpeptides being the most efficacious.

[0511] The same sera were also used to examine their effect on thebinding of IFNτ to its receptor.

[0512] The IFNτ binding assay was carried out as follows. Five μg ofIFNτ was iodinated for 2 minutes with 500 μCi of Na¹²⁵I (15 mCi/μg;Amersham Corporation, Arlington Heights, Ill.) in 25 μl of 0.5 Mpotassium phosphate buffer, pH 7.4, and 10 μl of chloramine-T (5 mg/ml)(Griggs, et al., 1992). The specific activity of the iodinated proteinwas 137 μCi/g. For binding assays, monolayers of MDBK cells were fixedwith paraformaldehyde and blocked with 5% nonfat dry milk. Cells wereincubated with 5 nM ¹²⁵I-IFNτ in phosphate buffered saline with 1% BSAfor 2 hours at 4° C. in the presence or absence of a 1:30 dilution ofsera containing antibodies raised against IFNτ peptides or theappropriate preimmune sera. Specific binding was assessed by incubationwith a 100-fold molar excess of unlabeled IFNτ. Specific binding of 36%was determined by competition with 500 nM unlabeled IFNτ. For example,total counts bound were 6850±133, and a 100-fold molar excess of OvIFNτproduced 4398±158 counts per minute. After incubation, the monolayerswere washed three times, solubilized with 1% sodium dodecyl sulfate, andthe radioactivity counted. Data from three replicate experiments arepresented in FIG. 18 as the mean percent reduction of OvIFNτspecificbinding produced by antipeptide antisera relative to the appropriatepreimmune sera+standard deviation. Significant differences were assessedby analysis of variance followed by Scheffe's F test.

[0513] The same sera (containing antibodies immunoreactive to OvIFNτ(1-37), OvIFNτ (62-92), and OvIFNτ (139-172)) were the most effectiveinhibitors of ¹²⁵1-IFNτ binding to its receptor on MDBK cells. The lackof effect of sera immunoreactive with other IFNτ-derived peptides wasnot a function of titer against OvIFNτ, since each sera had equal orgreater titer to their respective peptide relative to the threeinhibiting sera: similar results were obtained when sera reactivityagainst the whole OvIFNτ molecule was assessed by ELISA for each sera.

[0514] These peptides, although apparently binding to the interferonreceptor, did not in and of themselves elicit interferon-like effects inthe cells.

[0515] C. Anti-Proliferative Activity.

[0516] Functionally important sites for the antiproliferative activityof IFNτ were also examined using synthetic peptides (Table 10). Cellularproliferation was assayed as described above using MDBK cells. MDBKcells were cultured at 5×10⁵ cells/well in experiments 1 and 2 or 10×10⁵cells in experiment 3 and treated with medium alone, IFNτ at aconcentration of 300 units/ml and peptides at 1 mM for 48 hours.Duplicate wells were counted in each of three replicate experiments. Forstatistical analysis, data were normalized based on medium alone andassessed by analysis of variance followed by Least SignificantDifference multiplate comparison test (p>0.05). TABLE 10 PeptideInhibition of IFNτ Antiproliferative Activity Experiment 1 Experiment 2Experiment 3 Treatment Cell Count Via-bility Cell Count Via-bility CellCount Via-bility Medium alone 9.8 × 10⁵ 99% 13.0 × 10⁵ 96% 27.3 × 10⁵97% IFN^(τ) 5.0 × 10⁵ 98%  5.6 × 10⁵ 97%  8.3 × 10⁵ 97% IFN^(τ) +IFN^(τ) (1-37) 6.3 × 10⁵ 100% 10.6 × 10⁵ 98% 13.4 × 10⁵ 100% IFN^(τ) +IFN^(τ) (34-64) 5.3 × 10⁵ 96%  6.9 × 10⁵ 95% 16.0 × 10⁵ 98% IFN^(τ) +IFN^(τ) (62-92) 6.5 × 10⁵ 97%  9.2 × 10⁵ 93%  8.9 × 10⁵ 96% IFN^(τ) +IFN^(τ) (90-122) 5.9 × 10⁵ 100% 11.0 × 10⁵ 97% 19.6 × 10⁵ 98% IFN^(τ) +IFN^(τ) (119-150) 8.4 × 10⁵ 100% 13.2 × 10⁵ 96% 31.8 × 10⁵ 90% IFN^(τ) +IFN^(τ) (139-172) 5.1 × 10⁵ 100% 12.7 × 10⁵ 98% 18.9 × 10⁵ 98%

[0517] When proliferation of MDBK cells was monitored over a two-dayperiod, cell number increased roughly 2-fold with greater than 95%viability. Addition of 300 units/ml of OvIFNτ entirely eliminated cellproliferation without a decrease in cell viability. Ovine IFNτ (119-150)was the most effective inhibitor of IFNτ antiproliferative activity.

[0518] Antisera to IFNτ (119-150), which inhibited binding of OvIFNτ toreceptor, also reversed the OvIFNτ antiproliferative effect. Severalother peptides, notably IFNτ (139-172), reversed the OvIFNτantiproliferative effect, but to a lesser extent.

EXAMPLE 18 Further Analysis of the Cellular and Anti-Viral Effects ofIFNτ

[0519] A. HIV Anti-Viral Effects.

[0520] The antiviral effects of IFNτ against HIV were evaluated bytreating human PBMC cells with various amounts of either recombinantovine IFNτ (r-OvIFNτ) or recombinant human IFNα2a at the time ofinfection with HIV. IFNτ was present throughout the experiment. At day 7and day 14, p24 production was determined (by ELISA (Wang, et al .,1988, 1989) and compared to a zero drug control. The results of thisanalysis are presented in Table 11. TABLE 11 Amounts of % % DrugUnits/ml Inhibition Inhibition IFNα2a IFNτ Day 7 Day 14 10 26 58%, 48%91%, 91% 48%, 45% 88%, 59% 100 260 68%, 74% 94%, 91% 58%, 51% 82%, 70%1,000 2,600 89%, 86% 97%, 93% 65%, 68% 87%, 79% 10,000 26,000 90%, 86%99%, 99% 260,000 77%, 85% 77%, 96% 85%, 84% 96%, 86%

[0521] The data from these experiments support the concllusionl that, atrelatively low concentrations, IFNα2a and IFNτ are effective in reducingthe replication of HIV in human lymphocytes.

[0522] In Vitro Cytotoxicity Test in PBMC's

[0523] Human PBMC's were seeded at 5×10⁵ cells/ml. Cells were stimulatedat day 0 with 3 μg/ml PHA. Cells were treated with recombinant humanIFNα2A (at concentrations of 10, 100, 1,000 and 10,000 units/ml) andIFNτ (at concentrations of 2.6, 26, 260, 2,600, 26,000, 260,000, and2,600,000 units/ml) in 200 μl/wells (4 replicates of each concentrationusing 96 well flat bottom plates). Control cultures were given nointerferons. After 4 days of incubation, cells were pulsed for 9 hoursusing ³H-thymidine at 1 uCi/well. The cells were harvested and theincorporation of labeled thymidine into DNA was determined (FIG. 8).

[0524] No cytotoxicity was observed by measuring the uptake of thymidineat any concentration of IFNτ. However, rHuIFNα2 was toxic to cells at1,000 units/ml.

[0525] In a second experiment, the same human PBMC's were treated witheither IFNτ or human IFNα2A at concentrations of 100 units/ml or 10,000units/ml. After 3 days or 8 days of incubation, viable cells werecounted by flow cytometry. The results of this analysis are presented inTable 12. TABLE 12 Number of Viable Cells × 10,000 Treatment (units/ml)Day 3 Day 8 No Treatment 735 840 IFNτ 100 units/ml 745 860 IFNτ 10,000units/ml 695 910 IFNα2a 100 units/ml 635 750 IFNα2a 10,000 units/ml 680495

[0526] No cytotoxicity was observed in the cells treated with IFNτ.However, there was 10% cell death in IFNα2a treated cells at Day 3 and49% cell death at Day 8.

[0527] C. Inhibition of Hepatitis B Virus DNA Replication in Hepatocytes

[0528] The cell line used, HepG2-T14, is a human cell that was derivedfrom liver cells transfected with Hepatitis B Virus (HBV). The cell linesemi-stably produces HBV virus: over time the cell line's production ofHBV intracellular DNA and secreted virus decreases. In order to maximizeproduction of HBV DNA and virus, the cells are pre-treated with deAZA-C(5-azacytidine; Miyoshi, et al.) to induce production of the virus.Treatment was for 2-3 days and the amount of induction was about afactor of two.

[0529] The cells were then treated with either the IFNα and IFNτ atlevels of 0, 5,000, 10,000, 20,000 and 40,000 units per ml.

[0530] All levels of either IFNα or IFNτ reduced DNA production by abouta factor of 2 compared to the no drug control.

[0531] D. Inhibition of Hepatospecific Messenger RNA Production inHepatocytes

[0532] The hepatocyte cell line HepG2-T14 (described above) was examinedfor the effects of IFNα and IFNτ on hepatospecific mRNA production.Cells were incubated in concentrations of IFNα or IFNτ at 0, 5,000,10,000, 20,000, and 40,000 units per ml. The messenger RNAs for thehepatocyte specific proteins Apo E and Apo A1 were detected byhybridization analysis (Sambrook, et al.; Maniatis, et al.) using probesspecific for these two mRNA's (Shoulders, et al., and Wallis, et al.).

[0533] No reduction of mRNA production was seen for Apo E or Apo A1 mRNAproduction with up to 40,000 units of either IFNα or IFNτ. This resultsuggests that the reduction of viral DNA replication in previousexperiments was not due to the effects of IFNs on cellular house-keepingactivities; rather the reduction was likely due to specific inhibitionof viral replication in the host cells.

[0534] E. In Vitro Toxicity of IFNβ, IFNγ and IFNτ—L929 Cell Assay

[0535] The toxicity of IFN treatment was measured in vitro using themouse L929 cell line. L929 cells were treated with 6000 U/ml to 200,000U/ml of either OvIFNτ or MuIFNβ. The interferons were added at time zeroand the cells were incubated for 72 hours and stained with crystalviolet. The percentage of living cells was determined by measuring theabsorbance at 405 nm.

[0536] Exemplary data are shown in FIG. 21. Values are presented aspercent viability ± standard error in which 100 percent is equal to theviability of L929 cells treated with media alone. At 6000 U/ml,IFNβ-treated cells exhibited a 77.0±0.6% viability. Viability of L929cells decreased as the concentrations of IFNβ increased in adose-dependent manner. In contrast, L929 cells showed no decrease inviability at any of the IFNτ concentrations tested. These data indicatethat, unlike IFNβ, IFNτ lacks toxicity at high concentrations in vitro.

[0537] Taken together, the results summarized above demonstrate thatIFNτ is essentially non-toxic at concentrations at which IFNβ inducestoxicity both in vitro and in vivo.

[0538] In Vivo Toxicity of IFNβ, IFNγ and IFNτ—Cell Counts and WeightChanges

[0539] The effects of in vivo treatment with IFNτ, IFNβ and IFNα (10⁵U/injection) on total white blood cell (WBC), total lymphocyte countsand weight measurements in NZW mice were assessed as follows.Interferons (OvIFNτ, MuIFNβ, and MuIFNα) were injected intraperitoneally(i.p.) at a concentration of 10⁵ U in a total volume of 0.2 ml in PBSinto groups of New Zealand White (NZW) mice (Jackson Laboratories, BarHarbor, Me.). Three to four animals were included in each group. Whiteblood cell (WBC) counts were determined before injection and at selectedtimepoints thereafter (typically 12 and 24 hours) using a hemocytometerand standard techniques. Differential WBC counts were performed onWright-Giemsa stained blood smears. The Before injection, the weights ofthe animals ranged from 20 to 23 grams.

[0540] The results are summarized in Table 13, below. TABLE 13 IN VIVOTOXICITY OF INTERFERONS AS MEASURED BY WHITE BLOOD CELL COUNTS ANDPERCENT WEIGHT CHANGE % Weight Cell Count (Cell No. × 10³) % Change 24Before Injection 12 hr. after Injection Lymphocyte Hours after IFN TotalWBC Lymphocytes Total WBC Lymphocytes Depression Injection none 7.3 ±1.0 6.4 ± 0.7 8.0 ± 0.8 7.1 ± 0.7 0 +0.5 ± 0.7 τ 6.7 ± 0.7 5.9 ± 0.6 6.7± 0.5 5.8 ± 0.4 1.7 +1.3 ± 0.5 β 7.0 ± 1.4 6.0 ± 0.5 6.8 ± 0.8 4.1 ± 0.331.7 −20.0 ± 1.0  α 6.0 ± 0.8 5.2 ± 0.7 4.8 ± 0.5 2.3 ± 0.2 55.8 −8.5 ±2.0

[0541] No significant differences in WBC counts, lymphocyte counts orweight change were observed between IFNτ-treated and untreated mice. Incontrast, IFNβ-treated mice exhibited a 31.7% depression in lymphocytecounts 12 hours after injection, which continued for at least the next12 hours. IFNα-treated mice exhibited a 55.8% lymphocyte depression andsignificant weight loss 12 hours after injection. These data indicatethat, unlike IFNβ and IFNα, IFNτ lacks toxicity in vivo at the aboveconcentrations as evidenced by peripheral blood cell counts and weightmeasurements.

EXAMPLE 19 Isolation of Interferon-τ Fusion Protein

[0542] Sepharose 4B beads conjugated with anti-beta galactosidase ispurchased from Promega. The beads are packed in 2 ml column and washedsuccessively with phosphate-buffered saline with 0.02% sodium azide and10 ml TX buffer (10 mM Tris buffer, pH 7.4, 1% aprotinin).

[0543] The IFNτ coding sequence (e.g., SEQ ID NO:33, i.e., minus thenucleotides corresponding to the leader sequence) is cloned into thepolylinker site of lambda gt11. The IFNτ coding sequence is placedin-frame with the amino terminal β-galactosidase coding sequences inlambda gtll. Lysogens infected with gt11/IFNτ are used to inoculate 500ml of NZYDT broth. The culture is incubated at 32° C. with aeration toan O.D. of about 0.2 to 0.4, then brought to 43° C. quickly in a 43° C.water bath for 15 minutes to induce gt11 peptide synthesis, andincubated further at 37° C. for 1 hour. The cells are pelleted bycentrifugation, suspended in 10 ml of lysis buffer (10 mM Tris, pH 7.4containing 2% “TRITON X-100” and 1% aprotinin added just before use.

[0544] The resuspended cells are frozen in liquid nitrogen then thawed,resulting in substantially complete cell lysis. The lysate is treatedwith DNaseI to digest bacterial and phage DNA, as evidenced by a gradualloss of viscosity in the lysate. Non-solubilized material is removed bycentrifugation.,

[0545] The clarified lysate material is loaded on the Sepharose column,the ends of the column closed, and the column placed on a rotary shakerfor 2 hrs. at room temperature and 16 hours at 4° C. After the columnsettles, it is washed with 10 ml of TX buffer. The fused protein iseluted with 0.1 M carbonate/bicarbonate buffer, pH10. Typically, 14 mlof the elution buffer is passed through the column, and the fusionprotein is eluted in the first 4-6 ml of eluate.

[0546] The eluate containing the fusion protein is concentrated in“CENTRICON-30” cartridges (Amicon, Danvers, Mass.). The final proteinconcentrate is resuspended in, for example, 400 μl PBS buffer. Proteinpurity is analyzed by SDS-PAGE.

[0547] For polyclonal antibodies, the purified fused protein is injectedsubcutaneously in Freund's adjuvant in a rabbit. Approximately 1 mg offused protein is injected at days 0 and 21, and rabbit serum istypically collected at 6 and 8 weeks.

EXAMPLE 20 Preparation of Anti-IFNτ Antibody

[0548] A. Expression of Glutathione-S-Transferase Fusion Proteins

[0549] The IFNτ coding sequence (e.g., SEQ ID NO:33) is cloned into thepGEX vector (Boyer, et al.; Frangioni, et al.; Guan, et al.; Hakes, etal.; Smith, et al., 1988). The pGEX vector (Smith, et al.) was modifiedby insertion of a thrombin cleavage sequence in-frame with theglutathione-S-transferase protein (GST—sj26 coding sequence). Thisvector is designated pGEXthr. The IFNτ coding sequence is placedin-frame with the sj26-thrombin coding sequences (Guan, et al.; Hakes,et al.). The IFNτ coding sequence insert can be generated by thepolymerase chain reaction using PCR primers specific for the insert.

[0550] The IFNτ fragment is ligated to the linearized pGEXthr vector.The ligation mixture is transformed into E. coli and ampicillinresistant colonies are selected. Plasmids are isolated from theampicillin resistant colonies and analyzed by restriction enzymedigestion to identify clones containing the IFNτ insert (vectordesignated pGEXthr-IFNτ).

[0551]E. coli strain XL-I Blue is transformed with pGEXthr-IFNτ and isgrown at 37° C. overnight. DNA is prepared from randomly-pickedcolonies. The presence of the insert coding sequence is typicallyconfirmed by (i) restriction digest mapping, (ii) hybridizationscreening using labelled IFNτ probes (i.e., Southern analysis), or (iii)direct DNA sequence analysis.

[0552] B. Partial Purification of Fusion Proteins.

[0553] A pGEXthr-IFNτ clone is grown overnight. The overnight culture isdiluted 1:10 with LB medium containing ampicillin and grown for one hourat 37° C. Alternatively, the overnight culture is diluted 1:100 andgrown to OD of 0.5-1.0 before addition of IPTG(isopropylthio-β-galactoside). IPTG (GIBCO-BRL, Gaithersburg Md.) isadded to a final concentration of 0.2-0.5 mM for the induction ofprotein expression and the incubation is typically continued for 2-5hours, preferably 3.5 hours.

[0554] Bacterial cells are harvested by centrifugation and resuspendedin 1/100 culture volume of MTPBS (150 mM NaCl, 16 mM Na₂HPO₄, 4 mMNaH₂PO₄). Cells are lysed by lysozyme, sonication or French press, andlysates cleared of cellular debris by centrifugation.

[0555] An aliquot of the supernatant obtained from IPTG-induced culturesof pGEXthr-IFNτ-containing cells and an aliquot of the supernatantobtained from IPTG-induced cultures of pGEXthr-vector alone are analyzedby SDS-polyacrylamide gel electrophoresis followed by Western blotting,as described below.

[0556] If necessary, the extracts can be concentrated by ultrafiltrationusing, for example, a “CENTRICON 10” filter.

[0557] Alternatively, the fusion proteins are partially purified over aglutathione agarose affinity column as described in detail by Smith, etal. In this method, 100 ml cultures are grown overnight. The culturesare diluted to 1 liter, and the cells grown another hour at 37° C.Expression of the fusion proteins is induced using IPTG. The inducedcultures are grown at 37° C. for 3.5 hours. Cells are harvested and asonicator used to lyse the cells. Cellular debris is pelleted and theclear lysate loaded onto a glutathione “SEPHAROSE” column. The column iswashed with several column volumes. The fusion protein is eluted fromthe affinity column with reduced glutathione and dialyzed. The IFNτ canbe liberated from the hybrid protein by treatment with thrombin. Thesj26 and IFNτ fragments of the hybrid protein can then be separated bysize fractionation over columns or on gels.

[0558] Alternatively, the IFNτ portion of the hybrid protein is releasedfrom the column by treatment with thrombin (Guan, et al.; Hakes, etal.).

[0559] C. Antibodies Against the Fusion Protein.

[0560] The purified Sj26/IFNτ fused protein is injected subcutaneouslyin Freund's adjuvant in a rabbit. Approximately 1 mg of fused protein isinjected at days 0 and 21, and rabbit serum is typically collected at 6and 8 weeks. A second rabbit is similarly immunized with purified Sj26protein obtained from control bacterial lysate.

[0561] Minilysates from the following bacterial cultures are prepared:(1) KM392 cells infected with pGEXthr and pGEXthr containing the IFNτinsert; and (2) cells infected with lambda gt11 containing the IFNτinsert. The minilysates and a commercial source β-galactosidase arefractionated by SDS-PAGE, and the bands transferred to nitrocellulosefilters for Western blotting (Sambrook, et al.; Ausubel, et al.).

[0562] Summarizing the expected results, serum from control (Sj26)rabbits is immunoreactive with each of the Sj26 and Sj26 fused proteinantigens. Serum from the animal immunized with Sj26/IFNτ fused proteinis reactive with all Sj-26 and beta-gal fusion proteins containing IFNτcoding sequences, indicating the presence of specific immunoreactionwith the IFNτ antigen. None of the sera are expected to beimmunoreactive with beta-galactosidase.

[0563] Anti-IFNτ antibody present in the sera from the animal immunizedwith the Sj26/IFNτ is purified by affinity chromatography (usingimmobilized recombinantly produced IFNτ as ligand, essentially asdescribed above in Example 12 for the anti-beta-galactosidase antibody).

[0564] While the invention has been described with reference to specificmethods-and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES:44 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 516 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: Purification and Antiviral Activity (vi) ORIGINALSOURCE: (A) ORGANISM: Ovis aries (B) STRAIN: Domestic (D) DEVELOPMENTALSTAGE: Blastula (blastocyst) (F) TISSUE TYPE: Trophectoderm (G) CELLTYPE: Mononuclear trophectoderm cells (vii) IMMEDIATE SOURCE: (B) CLONE:Cloning and Expression in Saccharomyces cerevisiae of a Synthetic Genefor the Type I Trophoblast Interferon Ovine Trophoblast (viii) POSITIONIN GENOME: (C) UNITS: bp (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:1..516 (x) PUBLICATION INFORMATION: (A) AUTHORS: Ott, Troy L Van Heeke,Gino Johnson, Howard M Bazer, Fuller W (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 1: TGC TAC CTG TCG CGA AAA CTG ATG CTG GAC GCT CGA GAA AAT TTA AAA48 Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala Arg Glu Asn Leu Lys 1 510 15 CTG CTG GAC CGT ATG AAT CGA TTG TCT CCG CAC AGC TGC CTG CAA GAC 96Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu Gln Asp 20 25 30CGG AAA GAC TTC GGT CTG CCG CAG GAA ATG GTT GAA GGT GAC CAA CTG 144 ArgLys Asp Phe Gly Leu Pro Gln Glu Met Val Glu Gly Asp Gln Leu 35 40 45 CAAAAA GAC CAA GCT TTC CCG GTA CTG TAT GAA ATG CTG CAG CAG TCT 192 Gln LysAsp Gln Ala Phe Pro Val Leu Tyr Glu Met Leu Gln Gln Ser 50 55 60 TTC AACCTG TTC TAC ACT GAA CAT TCT TCG GCC GCT TGG GAC ACT ACT 240 Phe Asn LeuPhe Tyr Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 CTT CTAGAA CAA CTG TGC ACT GGT CTG CAA CAG CAA CTG GAC CAT CTG 288 Leu Leu GluGln Leu Cys Thr Gly Leu Gln Gln Gln Leu Asp His Leu 85 90 95 GAC ACT TGCCGT GGC CAG GTT ATG GGT GAA GAA GAC TCT GAA CTG GGT 336 Asp Thr Cys ArgGly Gln Val Met Gly Glu Glu Asp Ser Glu Leu Gly 100 105 110 AAC ATG GATCCG ATC GTT ACT GTT AAA AAA TAT TTC CAG GGT ATC TAC 384 Asn Met Asp ProIle Val Thr Val Lys Lys Tyr Phe Gln Gly Ile Tyr 115 120 125 GAC TAC CTGCAG GAA AAA GGT TAC TCT GAC TGC GCT TGG GAA ATC GTA 432 Asp Tyr Leu GlnGlu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val 130 135 140 CGC GTT GAAATG ATG CGG GCC CTG ACT GTG TCG ACT ACT CTG CAA AAA 480 Arg Val Glu MetMet Arg Ala Leu Thr Val Ser Thr Thr Leu Gln Lys 145 150 155 160 CGG TTAACT AAA ATG GGT GGT GAC CTG AAT TCT CCG 516 Arg Leu Thr Lys Met Gly GlyAsp Leu Asn Ser Pro 165 170 (2) INFORMATION FOR SEQ ID NO: 2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 172 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINALSOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence of a mature OvIFNtauprotein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Cys Tyr Leu Ser Arg LysLeu Met Leu Asp Ala Arg Glu Asn Leu Lys 1 5 10 15 Leu Leu Asp Arg MetAsn Arg Leu Ser Pro His Ser Cys Leu Gln Asp 20 25 30 Arg Lys Asp Phe GlyLeu Pro Gln Glu Met Val Glu Gly Asp Gln Leu 35 40 45 Gln Lys Asp Gln AlaPhe Pro Val Leu Tyr Glu Met Leu Gln Gln Ser 50 55 60 Phe Asn Leu Phe TyrThr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 Leu Leu Glu GlnLeu Cys Thr Gly Leu Gln Gln Gln Leu Asp His Leu 85 90 95 Asp Thr Cys ArgGly Gln Val Met Gly Glu Glu Asp Ser Glu Leu Gly 100 105 110 Asn Met AspPro Ile Val Thr Val Lys Lys Tyr Phe Gln Gly Ile Tyr 115 120 125 Asp TyrLeu Gln Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val 130 135 140 ArgVal Glu Met Met Arg Ala Leu Thr Val Ser Thr Thr Leu Gln Lys 145 150 155160 Arg Leu Thr Lys Met Gly Gly Asp Leu Asn Ser Pro 165 170 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:516 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: synthetic nucleotide sequence encoding a maturehuman interferon-tau protein, HuIFNtau1. (xi) SEQUENCE DESCRIPTION: SEQID NO: 3: TGTGACTTGT CTCAAAACCA CGTTTTGGTT GGTAGAAAGA ACTTAAGACTACTAGACGAA 60 ATGAGACGTC TATCTCCACG CTTCTGTCTA CAAGACAGAA AGGACTTCGCTTTGCCTCAG 120 GAAATGGTTG AAGGTGGCCA ACTACAAGAA GCTCAAGCGA TATCTGTTTTGCACGAAATG 180 TTGCAACAAA GCTTCAACTT GTTCCACACC GAACACTCTT CGGCCGCTTGGGACACCACC 240 TTGTTGGAAC AGCTCAGAAC CGGTTTGCAC CAACAATTGG ACAACTTGGATGCATGTTTG 300 GGTCAAGTTA TGGGTGAAGA AGACTCTGCT CTCGGGAGAA CCGGTCCAACGCTAGCTTTG 360 AAGAGATACT TCCAAGGTAT CCACGTTTAC TTGAAGGAAA AGGGTTACTCTGACTGTGCT 420 TGGGAAACCG TGCGTCTAGA AATCATGCGT AGCTTCTCTT CTTTGATCAGCTTGCAAGAA 480 AGATTACGTA TGATGGACGG TGACTTGTCG AGCCCA 516 (2)INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:172 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: amino acid sequence for a mature HuIFNtau protein,HuIFNtau1. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Cys Asp Leu Ser GlnAsn His Val Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 15 Leu Leu Asp GluMet Arg Arg Leu Ser Pro Arg Phe Cys Leu Gln Asp 20 25 30 Arg Lys Asp PheAla Leu Pro Gln Glu Met Val Glu Gly Gly Gln Leu 35 40 45 Gln Glu Ala GlnAla Ile Ser Val Leu His Glu Met Leu Gln Gln Ser 50 55 60 Phe Asn Leu PheHis Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 Leu Leu GluGln Leu Arg Thr Gly Leu His Gln Gln Leu Asp Asn Leu 85 90 95 Asp Ala CysLeu Gly Gln Val Met Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110 Arg ThrGly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gln Gly Ile His 115 120 125 ValTyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val 130 135 140Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Leu Ile Ser Leu Gln Glu 145 150155 160 Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acidsequence of fragment 1-37 of SEQ ID NO:2 (xi) SEQUENCE DESCRIPTION: SEQID NO: 5: Cys Tyr Leu Ser Arg Lys Leu Met Leu Asp Ala Arg Glu Asn LeuLys 5 10 15 Leu Leu Asp Arg Met Asn Arg Leu Ser Pro His Ser Cys Leu GlnAsp 20 25 30 Arg Lys Asp Phe Gly 35 (2) INFORMATION FOR SEQ ID NO: 6:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINALSOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence of fragment 34-64 ofSEQ ID NO:2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Lys Asp Phe Gly LeuPro Gln Glu Met Val Glu Gly Asp Gln Leu Gln 5 10 15 Lys Asp Gln Ala PhePro Val Leu Tyr Glu Met Leu Gln Gln Ser 20 25 30 (2) INFORMATION FOR SEQID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence of fragment62-92 of SEQ ID NO:2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Gln GlnSer Phe Asn Leu Phe Tyr Thr Glu His Ser Ser Ala Ala Trp 5 10 15 Asp ThrThr Leu Leu Glu Gln Leu Cys Thr Gly Leu Gln Gln Gln 20 25 30 (2)INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acidsequence of fragment 90-122 of SEQ ID NO:2 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 8: Gln Gln Gln Leu Asp His Leu Asp Thr Cys Arg Gly Gln ValMet Gly 5 10 15 Glu Glu Asp Ser Glu Leu Gly Asn Met Asp Pro Ile Val ThrVal Lys 20 25 30 Lys (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: amino acid sequence of fragment 119-150 of SEQ IDNO:2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Thr Val Lys Lys Tyr PheGln Gly Ile Tyr Asp Tyr Leu Gln Glu Lys 5 10 15 Gly Tyr Ser Asp Cys AlaTrp Glu Ile Val Arg Val Glu Met Met Arg 20 25 30 (2) INFORMATION FOR SEQID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: amino acid sequence of fragment139-172 of SEQ ID NO:2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Cys AlaTrp Glu Ile Val Arg Val Glu Met Met Arg Ala Leu Thr Val 5 10 15 Ser ThrThr Leu Gln Lys Arg Leu Thr Lys Met Gly Gly Asp Leu Asn 20 25 30 Ser Pro(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: HuIFNtau1 Human Interferon Tau coding sequence witha leader sequence. (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..585(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG GCC TTC GTG CTC TCT CTACTC ATG GCC CTG GTG CTG GTC AGC TAC 48 Met Ala Phe Val Leu Ser Leu LeuMet Ala Leu Val Leu Val Ser Tyr 1 5 10 15 GGC CCA GGA GGA TCC CTG GGTTGT GAC CTG TCT CAG AAC CAC GTG CTG 96 Gly Pro Gly Gly Ser Leu Gly CysAsp Leu Ser Gln Asn His Val Leu 20 25 30 GTT GGC AGG AAG AAC CTC AGG CTCCTG GAC GAA ATG AGG AGA CTC TCC 144 Val Gly Arg Lys Asn Leu Arg Leu LeuAsp Glu Met Arg Arg Leu Ser 35 40 45 CCT CGC TTT TGT CTG CAG GAC AGA AAAGAC TTC GCT TTA CCC CAG GAA 192 Pro Arg Phe Cys Leu Gln Asp Arg Lys AspPhe Ala Leu Pro Gln Glu 50 55 60 ATG GTG GAG GGC GGC CAG CTC CAG GAG GCCCAG GCC ATC TCT GTG CTC 240 Met Val Glu Gly Gly Gln Leu Gln Glu Ala GlnAla Ile Ser Val Leu 65 70 75 80 CAT GAG ATG CTC CAG CAG AGC TTC AAC CTCTTC CAC ACA GAG CAC TCC 288 His Glu Met Leu Gln Gln Ser Phe Asn Leu PheHis Thr Glu His Ser 85 90 95 TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CAGCTC CGC ACT GGA CTC 336 Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gln LeuArg Thr Gly Leu 100 105 110 CAT CAG CAG CTG GAC AAC CTG GAT GCC TGC CTGGGG CAG GTG ATG GGA 384 His Gln Gln Leu Asp Asn Leu Asp Ala Cys Leu GlyGln Val Met Gly 115 120 125 GAG GAA GAC TCT GCC CTG GGA AGG ACG GGC CCCACC CTG GCT CTG AAG 432 Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro ThrLeu Ala Leu Lys 130 135 140 AGG TAC TTC CAG GGC ATC CAT GTC TAC CTG AAAGAG AAG GGA TAC AGC 480 Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Lys GluLys Gly Tyr Ser 145 150 155 160 GAC TGC GCC TGG GAA ACC GTC AGA CTG GAAATC ATG AGA TCC TTC TCT 528 Asp Cys Ala Trp Glu Thr Val Arg Leu Glu IleMet Arg Ser Phe Ser 165 170 175 TCA TTA ATC AGC TTG CAA GAA AGG TTA AGAATG ATG GAT GGA GAC CTG 576 Ser Leu Ile Ser Leu Gln Glu Arg Leu Arg MetMet Asp Gly Asp Leu 180 185 190 AGC TCA CCT TGA 588 Ser Ser Pro 195 (2)INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:195 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: predictedamino acid coding sequence of SEQ ID NO:11 (HuIFNtau1). (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 12: Met Ala Phe Val Leu Ser Leu Leu Met Ala LeuVal Leu Val Ser Tyr 1 5 10 15 Gly Pro Gly Gly Ser Leu Gly Cys Asp LeuSer Gln Asn His Val Leu 20 25 30 Val Gly Arg Lys Asn Leu Arg Leu Leu AspGlu Met Arg Arg Leu Ser 35 40 45 Pro Arg Phe Cys Leu Gln Asp Arg Lys AspPhe Ala Leu Pro Gln Glu 50 55 60 Met Val Glu Gly Gly Gln Leu Gln Glu AlaGln Ala Ile Ser Val Leu 65 70 75 80 His Glu Met Leu Gln Gln Ser Phe AsnLeu Phe His Thr Glu His Ser 85 90 95 Ser Ala Ala Trp Asp Thr Thr Leu LeuGlu Gln Leu Arg Thr Gly Leu 100 105 110 His Gln Gln Leu Asp Asn Leu AspAla Cys Leu Gly Gln Val Met Gly 115 120 125 Glu Glu Asp Ser Ala Leu GlyArg Thr Gly Pro Thr Leu Ala Leu Lys 130 135 140 Arg Tyr Phe Gln Gly IleHis Val Tyr Leu Lys Glu Lys Gly Tyr Ser 145 150 155 160 Asp Cys Ala TrpGlu Thr Val Arg Leu Glu Ile Met Arg Ser Phe Ser 165 170 175 Ser Leu IleSer Leu Gln Glu Arg Leu Arg Met Met Asp Gly Asp Leu 180 185 190 Ser SerPro 195 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 25 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (vi) ORIGINALSOURCE: (C) INDIVIDUAL ISOLATE: 25-mer synthetic oligonucleotide (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 13: CCTGTCTGCA GGACAGAAAA GACTT 25 (2)INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:25 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:linear (ii) MOLECULE TYPE: DNA (synthetic) (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: 25-mer synthetic oligonucleotide (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 14: TCTGAATTCT GACGATTTCC CAGGC 25 (2)INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:37 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: Amino acid sequence of fragment 1-37 of SEQ ID NO:4(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Cys Asp Leu Ser Gln Asn HisVal Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 15 Leu Leu Asp Glu Met ArgArg Leu Ser Pro Arg Phe Cys Leu Gln Asp 20 25 30 Arg Lys Asp Phe Ala 35(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: Amino acid sequence of fragment 34-64 of SEQ ID NO:4(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: Lys Asp Phe Ala Leu Pro GlnGlu Met Val Glu Gly Gly Gln Leu Gln 1 5 10 15 Glu Ala Gln Ala Ile SerVal Leu His Glu Met Leu Gln Gln Ser 20 25 30 (2) INFORMATION FOR SEQ IDNO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Aminoacid sequence of fragment 62-92 of SEQ ID NO:4 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 17: Gln Gln Ser Phe Asn Leu Phe His Thr Glu HisSer Ser Ala Ala Trp 1 5 10 15 Asp Thr Thr Leu Leu Glu Gln Leu Arg ThrGly Leu His Gln Gln 20 25 30 (2) INFORMATION FOR SEQ ID NO: 18: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Aminoacid sequence of fragment 90-122 of SEQ ID NO:4 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 18: His Gln Gln Leu Asp Asn Leu Asp Ala Cys LeuGly Gln Val Met Gly 1 5 10 15 Glu Glu Asp Ser Ala Leu Gly Arg Thr GlyPro Thr Leu Ala Leu Lys 20 25 30 Arg (2) INFORMATION FOR SEQ ID NO: 19:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Aminoacid sequence of fragment 119-150 of SEQ ID NO:4 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 19: Ala Leu Lys Arg Tyr Phe Gln Gly Ile His ValTyr Leu Lys Glu Lys 1 5 10 15 Gly Tyr Ser Asp Cys Ala Trp Glu Thr ValArg Leu Glu Ile Met Arg 20 25 30 (2) INFORMATION FOR SEQ ID NO: 20: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Aminoacid sequence of fragment 139-172 of SEQ ID NO:4 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 20: Cys Ala Trp Glu Thr Val Arg Leu Glu Ile MetArg Ser Phe Ser Ser 1 5 10 15 Leu Ile Ser Leu Gln Glu Arg Leu Arg MetMet Asp Gly Asp Leu Ser 20 25 30 Ser Pro (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 299 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: HuIFNtau6 (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 2..298 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: C CAG GAG ATG GTG GAG GGC GGC CAG CTC CAG GAG GCC CAG GCC ATC 46 GlnGlu Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gln Ala Ile 1 5 10 15 TCTGTG CTC CAC AAG ATG CTC CAG CAG AGC TTC AAC CTC TTC CAC ACA 94 Ser ValLeu His Lys Met Leu Gln Gln Ser Phe Asn Leu Phe His Thr 20 25 30 GAG CGCTCC TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CAG CTC CGC 142 Glu Arg SerSer Ala Ala Trp Asp Thr Thr Leu Leu Glu Gln Leu Arg 35 40 45 ACT GGA CTCCAT CAG CAG CTG GAT GAC CTG GAC GCC TGC CTG GGG CAG 190 Thr Gly Leu HisGln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly Gln 50 55 60 GTG ACG GGA GAGGAA GAC TCT GCC CTG GGA AGG ACG GGC CCC ACC CTG 238 Val Thr Gly Glu GluAsp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu 65 70 75 GCC GTG AAG AGC TACTTC CAG GGC ATC CAT ATC TAC CTG CAA GAG AAG 286 Ala Val Lys Ser Tyr PheGln Gly Ile His Ile Tyr Leu Gln Glu Lys 80 85 90 95 GGA TAC AGC GAC T299 Gly Tyr Ser Asp (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 99 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: predicted amino acid coding sequence of SEQ ID NO:21(HuIFNtau6). (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Gln Glu Met ValGlu Gly Gly Gln Leu Gln Glu Ala Gln Ala Ile Ser 1 5 10 15 Val Leu HisLys Met Leu Gln Gln Ser Phe Asn Leu Phe His Thr Glu 20 25 30 Arg Ser SerAla Ala Trp Asp Thr Thr Leu Leu Glu Gln Leu Arg Thr 35 40 45 Gly Leu HisGln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly Gln Val 50 55 60 Thr Gly GluGlu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala 65 70 75 80 Val LysSer Tyr Phe Gln Gly Ile His Ile Tyr Leu Gln Glu Lys Gly 85 90 95 Tyr SerAsp (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 288 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: HuIFNtau7 (ix) FEATURE: (A) NAME/KEY: CDS (B)LOCATION: 2..286 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: C CAG GAG ATGGTG GAG GTC AGC CAG TTC CAG GAG GCC CAG GCC ATT 46 Gln Glu Met Val GluVal Ser Gln Phe Gln Glu Ala Gln Ala Ile 1 5 10 15 TCT GTG CTC CAT GAGATG CTC CAG CAG AGC TTC AAC CTC TTC CAC AAA 94 Ser Val Leu His Glu MetLeu Gln Gln Ser Phe Asn Leu Phe His Lys 20 25 30 GAG CGC TCC TCT GCT GCCTGG GAC ACT ACC CTC CTG GAG CAG CTC CTC 142 Glu Arg Ser Ser Ala Ala TrpAsp Thr Thr Leu Leu Glu Gln Leu Leu 35 40 45 ACT GGA CTC CAT CAG CAG CTGGAT GAC CTG GAT GCC TGT CTG GGG CAG 190 Thr Gly Leu His Gln Gln Leu AspAsp Leu Asp Ala Cys Leu Gly Gln 50 55 60 TTG ACT GGA GAG GAA GAC TCT GCCCTG GGA AGG ACG GGC CCC ACC CTG 238 Leu Thr Gly Glu Glu Asp Ser Ala LeuGly Arg Thr Gly Pro Thr Leu 65 70 75 GCC GTG AAG AGC TAC TTC CAG GGC ATCCAT GTC TAC CTG CAA GAG AAG 286 Ala Val Lys Ser Tyr Phe Gln Gly Ile HisVal Tyr Leu Gln Glu Lys 80 85 90 95 GG 288 (2) INFORMATION FOR SEQ IDNO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: predicted amino acid codingsequence of SEQ ID NO:23 (HuIFNtau7). (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 24: Gln Glu Met Val Glu Val Ser Gln Phe Gln Glu Ala Gln Ala Ile Ser1 5 10 15 Val Leu His Glu Met Leu Gln Gln Ser Phe Asn Leu Phe His LysGlu 20 25 30 Arg Ser Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gln Leu LeuThr 35 40 45 Gly Leu His Gln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly GlnLeu 50 55 60 Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr LeuAla 65 70 75 80 Val Lys Ser Tyr Phe Gln Gly Ile His Val Tyr Leu Gln GluLys 85 90 95 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 307 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA tomRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:(C) INDIVIDUAL ISOLATE: HuIFNtau4 (ix) FEATURE: (A) NAME/KEY: CDS (B)LOCATION: 2..307 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: C CAG GAG ATGGTG GAG GGT GGC CAG CTC CAG GAG GCC CAG GCC ATC 46 Gln Glu Met Val GluGly Gly Gln Leu Gln Glu Ala Gln Ala Ile 1 5 10 15 TCT GTG CTC CAC GAGATG CTC CAG CAG AGC TTC AAC CTC TTC CAC ACA 94 Ser Val Leu His Glu MetLeu Gln Gln Ser Phe Asn Leu Phe His Thr 20 25 30 GAG CAC TCC TCT GCT GCCTGG GAC ACC ACC CTC CTG GAG CAG CTC CGC 142 Glu His Ser Ser Ala Ala TrpAsp Thr Thr Leu Leu Glu Gln Leu Arg 35 40 45 ACT GGA CTC CAT CAG CAG CTGGAT GAC CTG GAT GCC TGC CTG GGG CAG 190 Thr Gly Leu His Gln Gln Leu AspAsp Leu Asp Ala Cys Leu Gly Gln 50 55 60 GTG ACG GGA GAG GAA GAC TCT GCCCTG GGA AGG ACG GGC CCC ACC CTG 238 Val Thr Gly Glu Glu Asp Ser Ala LeuGly Arg Thr Gly Pro Thr Leu 65 70 75 GCC ATG AAG ACG TAT TTC CAG GGC ATCCAT GTC TAC CTG AAA GAG AAG 286 Ala Met Lys Thr Tyr Phe Gln Gly Ile HisVal Tyr Leu Lys Glu Lys 80 85 90 95 GGA TAT AGT GAC TGC GCC TGG 307 GlyTyr Ser Asp Cys Ala Trp 100 (2) INFORMATION FOR SEQ ID NO: 26: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 102 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINALSOURCE: (C) INDIVIDUAL ISOLATE: predicted amino acid coding sequence ofSEQ ID NO:25 (HuIFNtau4). (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: GlnGlu Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gln Ala Ile Ser 1 5 10 15Val Leu His Glu Met Leu Gln Gln Ser Phe Asn Leu Phe His Thr Glu 20 25 30His Ser Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gln Leu Arg Thr 35 40 45Gly Leu His Gln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly Gln Val 50 55 60Thr Gly Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu Ala 65 70 7580 Met Lys Thr Tyr Phe Gln Gly Ile His Val Tyr Leu Lys Glu Lys Gly 85 9095 Tyr Ser Asp Cys Ala Trp 100 (2) INFORMATION FOR SEQ ID NO: 27: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 294 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:cDNA to mRNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (C) INDIVIDUAL ISOLATE: HuIFNtau5 (ix) FEATURE: (A) NAME/KEY:CDS (B) LOCATION: 2..292 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: C CAGGAG ATG GTG GAG GGT GGC CAG CTC CAG GAG GCC CAG GCC ATC 46 Gln Glu MetVal Glu Gly Gly Gln Leu Gln Glu Ala Gln Ala Ile 1 5 10 15 TCT GTG CTCCAC GAG ATG CTC CAG CAG AGC TTC AAC CTC TTC CAC ACA 94 Ser Val Leu HisGlu Met Leu Gln Gln Ser Phe Asn Leu Phe His Thr 20 25 30 GAG CAC TCC TCTGCT GCC TGG GAC ACC ACC CTC CTG GAG CAG CTC CGC 142 Glu His Ser Ser AlaAla Trp Asp Thr Thr Leu Leu Glu Gln Leu Arg 35 40 45 ACT GGA CTC CAT CAGCAG CTG GAT GAC CTG GAT GCC TGC CTG GGG CAG 190 Thr Gly Leu His Gln GlnLeu Asp Asp Leu Asp Ala Cys Leu Gly Gln 50 55 60 GTG ACG GGA GAG GAA GACTCT GCC CTG GGA AGG ACG GGC CCC ACC CTG 238 Val Thr Gly Glu Glu Asp SerAla Leu Gly Arg Thr Gly Pro Thr Leu 65 70 75 GCC ATG AAG ACG TAT TTC CAGGGC ATC CAT GTC TAC CTG AAA GAG AAG 286 Ala Met Lys Thr Tyr Phe Gln GlyIle His Val Tyr Leu Lys Glu Lys 80 85 90 95 GGA TAT AG 294 Gly Tyr (2)INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:97 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: predictedamino acid coding sequence of SEQ ID NO:27 (HuIFNtau5). (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 28: Gln Glu Met Val Glu Gly Gly Gln Leu Gln GluAla Gln Ala Ile Ser 1 5 10 15 Val Leu His Glu Met Leu Gln Gln Ser PheAsn Leu Phe His Thr Glu 20 25 30 His Ser Ser Ala Ala Trp Asp Thr Thr LeuLeu Glu Gln Leu Arg Thr 35 40 45 Gly Leu His Gln Gln Leu Asp Asp Leu AspAla Cys Leu Gly Gln Val 50 55 60 Thr Gly Glu Glu Asp Ser Ala Leu Gly ArgThr Gly Pro Thr Leu Ala 65 70 75 80 Met Lys Thr Tyr Phe Gln Gly Ile HisVal Tyr Leu Lys Glu Lys Gly 85 90 95 Tyr (2) INFORMATION FOR SEQ ID NO:29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 516 base pairs (B) TYPE:nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULETYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: HuIFNtau2 (ix) FEATURE: (A)NAME/KEY: CDS (B) LOCATION: 1..516 (ix) FEATURE: (A) NAME/KEY:Modified-site (B) LOCATION: 115-117 (D) OTHER INFORMATION: /note= “toallow expression of the encoded protein this site can be modified toencode an amino acid, e.g., Gln” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GAC CTG TCT CAG AAC CAC GTG CTG GTT GGC AGG AAG AAC CTC AGG CTC 48Asp Leu Ser Gln Asn His Val Leu Val Gly Arg Lys Asn Leu Arg Leu 1 5 1015 CTG GAC CAA ATG AGG AGA CTC TCC CCT CGC TTT TGT CTG CAG GAC AGA 96Leu Asp Gln Met Arg Arg Leu Ser Pro Arg Phe Cys Leu Gln Asp Arg 20 25 30AAA GAC TTC GCT TTA CCC TAG GAA ATG GTG GAG GGC GGC CAG CTC CAG 144 LysAsp Phe Ala Leu Pro Glu Met Val Glu Gly Gly Gln Leu Gln 35 40 45 GAG GCCCAG GCC ATC TCT GTG CTC CAT GAG ATG CTC CAG CAG AGC TTC 192 Glu Ala GlnAla Ile Ser Val Leu His Glu Met Leu Gln Gln Ser Phe 50 55 60 AAC CTC TTCCAC ACA GAG CAC TCC TCT GCT GCC TGG GAC ACC ACC CTC 240 Asn Leu Phe HisThr Glu His Ser Ser Ala Ala Trp Asp Thr Thr Leu 65 70 75 80 CTG GAG CAGCTC CGC ACT GGA CTC CAT CAG CAG CTG GAC AAC CTG GAT 288 Leu Glu Gln LeuArg Thr Gly Leu His Gln Gln Leu Asp Asn Leu Asp 85 90 95 GCC TGC CTG GGGCAG GTG ATG GGA GAG GAA GAC TCT GCC CTG GGA AGG 336 Ala Cys Leu Gly GlnVal Met Gly Glu Glu Asp Ser Ala Leu Gly Arg 100 105 110 ACG GGC CCC ACCCTG GCT CTG AAG AGG TAC TTC CAG GGC ATC CAT GTC 384 Thr Gly Pro Thr LeuAla Leu Lys Arg Tyr Phe Gln Gly Ile His Val 115 120 125 TAC CTG AAA GAGAAG GGA TAC AGC GAC TGC GCC TGG GAA ACC GTC AGA 432 Tyr Leu Lys Glu LysGly Tyr Ser Asp Cys Ala Trp Glu Thr Val Arg 130 135 140 GTG GAA ATC ATGAGA TCC TTC TCT TCA TTA ATC AGC TTG CAA GAA AGG 480 Val Glu Ile Met ArgSer Phe Ser Ser Leu Ile Ser Leu Gln Glu Arg 145 150 155 160 TTA AGA ATGATG GAT GGA GAC CTG AGC TCA CCT TGA 516 Leu Arg Met Met Asp Gly Asp LeuSer Ser Pro 165 170 (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 171 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: predicted amino acid coding sequence of SEQ ID NO:29(HuIFNtau2). (ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 39(D) OTHER INFORMATION: /note= “where Xaa a selected amino acid, forexample, Gln” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Asp Leu Ser GlnAsn His Val Leu Val Gly Arg Lys Asn Leu Arg Leu 1 5 10 15 Leu Asp GlnMet Arg Arg Leu Ser Pro Arg Phe Cys Leu Gln Asp Arg 20 25 30 Lys Asp PheAla Leu Pro Xaa Glu Met Val Glu Gly Gly Gln Leu Gln 35 40 45 Glu Ala GlnAla Ile Ser Val Leu His Glu Met Leu Gln Gln Ser Phe 50 55 60 Asn Leu PheHis Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr Leu 65 70 75 80 Leu GluGln Leu Arg Thr Gly Leu His Gln Gln Leu Asp Asn Leu Asp 85 90 95 Ala CysLeu Gly Gln Val Met Gly Glu Glu Asp Ser Ala Leu Gly Arg 100 105 110 ThrGly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gln Gly Ile His Val 115 120 125Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val Arg 130 135140 Val Glu Ile Met Arg Ser Phe Ser Ser Leu Ile Ser Leu Gln Glu Arg 145150 155 160 Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170 (2)INFORMATION FOR SEQ ID NO: 31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:588 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE:HuIFNtau3 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..588 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 31: ATG GCC TTC GTG CTC TCT CTA CTC ATGGCC CTG GTG CTG GTC AGC TAC 48 Met Ala Phe Val Leu Ser Leu Leu Met AlaLeu Val Leu Val Ser Tyr 1 5 10 15 GGC CCG GGA GGA TCC CTG CGG TGT GACCTG TCT CAG AAC CAC GTG CTG 96 Gly Pro Gly Gly Ser Leu Arg Cys Asp LeuSer Gln Asn His Val Leu 20 25 30 GTT GGC AGC CAG AAC CTC AGG CTC CTG GGCCAA ATG AGG AGA CTC TCC 144 Val Gly Ser Gln Asn Leu Arg Leu Leu Gly GlnMet Arg Arg Leu Ser 35 40 45 CTT CGC TTC TGT CTG CAG GAC AGA AAA GAC TTCGCT TTC CCC CAG GAG 192 Leu Arg Phe Cys Leu Gln Asp Arg Lys Asp Phe AlaPhe Pro Gln Glu 50 55 60 ATG GTG GAG GGT GGC CAG CTC CAG GAG GCC CAG GCCATC TCT GTG CTC 240 Met Val Glu Gly Gly Gln Leu Gln Glu Ala Gln Ala IleSer Val Leu 65 70 75 80 CAC GAG ATG CTC CAG CAG AGC TTC AAC CTC TTC CACACA GAG CAC TCC 288 His Glu Met Leu Gln Gln Ser Phe Asn Leu Phe His ThrGlu His Ser 85 90 95 TCT GCT GCC TGG GAC ACC ACC CTC CTG GAG CAG CTC CGCACT GGA CTC 336 Ser Ala Ala Trp Asp Thr Thr Leu Leu Glu Gln Leu Arg ThrGly Leu 100 105 110 CAT CAG CAG CTG GAT GAC CTG GAT GCC TGC CTG GGG CAGGTG ACG GGA 384 His Gln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly Gln ValThr Gly 115 120 125 GAG GAA GAC TCT GCC CTG GGA AGA ACG GGC CCC ACC CTGGCC ATG AAG 432 Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr Leu AlaMet Lys 130 135 140 AGG TAT TTC CAG GGC ATC CAT GTC TAC CTG AAA GAG AAGGGA TAT AGT 480 Arg Tyr Phe Gln Gly Ile His Val Tyr Leu Lys Glu Lys GlyTyr Ser 145 150 155 160 GAC TGC GCC TGG GAA ATT GTC AGA CTG GAA ATC ATGAGA TCC TTG TCT 528 Asp Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met ArgSer Leu Ser 165 170 175 TCA TCA ACC AGC TTG CAC AAA AGG TTA AGA ATG ATGGAT GGA GAC CTG 576 Ser Ser Thr Ser Leu His Lys Arg Leu Arg Met Met AspGly Asp Leu 180 185 190 AGC TCA CCT TGA 588 Ser Ser Pro 195 (2)INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:195 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: predictedamino acid coding sequence of SEQ ID NO:31 (HuIFNtau3) (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 32: Met Ala Phe Val Leu Ser Leu Leu Met Ala LeuVal Leu Val Ser Tyr 1 5 10 15 Gly Pro Gly Gly Ser Leu Arg Cys Asp LeuSer Gln Asn His Val Leu 20 25 30 Val Gly Ser Gln Asn Leu Arg Leu Leu GlyGln Met Arg Arg Leu Ser 35 40 45 Leu Arg Phe Cys Leu Gln Asp Arg Lys AspPhe Ala Phe Pro Gln Glu 50 55 60 Met Val Glu Gly Gly Gln Leu Gln Glu AlaGln Ala Ile Ser Val Leu 65 70 75 80 His Glu Met Leu Gln Gln Ser Phe AsnLeu Phe His Thr Glu His Ser 85 90 95 Ser Ala Ala Trp Asp Thr Thr Leu LeuGlu Gln Leu Arg Thr Gly Leu 100 105 110 His Gln Gln Leu Asp Asp Leu AspAla Cys Leu Gly Gln Val Thr Gly 115 120 125 Glu Glu Asp Ser Ala Leu GlyArg Thr Gly Pro Thr Leu Ala Met Lys 130 135 140 Arg Tyr Phe Gln Gly IleHis Val Tyr Leu Lys Glu Lys Gly Tyr Ser 145 150 155 160 Asp Cys Ala TrpGlu Ile Val Arg Leu Glu Ile Met Arg Ser Leu Ser 165 170 175 Ser Ser ThrSer Leu His Lys Arg Leu Arg Met Met Asp Gly Asp Leu 180 185 190 Ser SerPro 195 (2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 518 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: HuIFNtau3, mature no leader sequence (ix) FEATURE:(A) NAME/KEY: CDS (B) LOCATION: 1..518 (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 33: TGT GAC CTG TCT CAG AAC CAC GTG CTG GTT GGC AGC CAG AAC CTC AGG48 Cys Asp Leu Ser Gln Asn His Val Leu Val Gly Ser Gln Asn Leu Arg 1 510 15 CTC CTG GGC CAA ATG AGG AGA CTC TCC CTT CGC TTC TGT CTG CAG GAC 96Leu Leu Gly Gln Met Arg Arg Leu Ser Leu Arg Phe Cys Leu Gln Asp 20 25 30AGA AAA GAC TTC GCT TTC CCC CAG GAG ATG GTG GAG GGT GGC CAG CTC 144 ArgLys Asp Phe Ala Phe Pro Gln Glu Met Val Glu Gly Gly Gln Leu 35 40 45 CAGGAG GCC CAG GCC ATC TCT GTG CTC CAC GAG ATG CTC CAG CAG AGC 192 Gln GluAla Gln Ala Ile Ser Val Leu His Glu Met Leu Gln Gln Ser 50 55 60 TTC AACCTC TTC CAC ACA GAG CAC TCC TCT GCT GCC TGG GAC ACC ACC 240 Phe Asn LeuPhe His Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 CTC CTGGAG CAG CTC CGC ACT GGA CTC CAT CAG CAG CTG GAT GAC CTG 288 Leu Leu GluGln Leu Arg Thr Gly Leu His Gln Gln Leu Asp Asp Leu 85 90 95 GAT GCC TGCCTG GGG CAG GTG ACG GGA GAG GAA GAC TCT GCC CTG GGA 336 Asp Ala Cys LeuGly Gln Val Thr Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110 AGA ACG GGCCCC ACC CTG GCC ATG AAG AGG TAT TTC CAG GGC ATC CAT 384 Arg Thr Gly ProThr Leu Ala Met Lys Arg Tyr Phe Gln Gly Ile His 115 120 125 GTC TAC CTGAAA GAG AAG GGA TAT AGT GAC TGC GCC TGG GAA ATT GTC 432 Val Tyr Leu LysGlu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Ile Val 130 135 140 AGA CTG GAAATC ATG AGA TCC TTG TCT TCA TCA ACC AGC TTG CAC AAA 480 Arg Leu Glu IleMet Arg Ser Leu Ser Ser Ser Thr Ser Leu His Lys 145 150 155 160 AGG TTAAGA ATG ATG GAT GGA GAC CTG AGC TCA CCT TG 518 Arg Leu Arg Met Met AspGly Asp Leu Ser Ser Pro 165 170 (2) INFORMATION FOR SEQ ID NO: 34: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 172 amino acids (B) TYPE: aminoacid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 34: Cys Asp Leu Ser Gln Asn His Val Leu Val GlySer Gln Asn Leu Arg 1 5 10 15 Leu Leu Gly Gln Met Arg Arg Leu Ser LeuArg Phe Cys Leu Gln Asp 20 25 30 Arg Lys Asp Phe Ala Phe Pro Gln Glu MetVal Glu Gly Gly Gln Leu 35 40 45 Gln Glu Ala Gln Ala Ile Ser Val Leu HisGlu Met Leu Gln Gln Ser 50 55 60 Phe Asn Leu Phe His Thr Glu His Ser SerAla Ala Trp Asp Thr Thr 65 70 75 80 Leu Leu Glu Gln Leu Arg Thr Gly LeuHis Gln Gln Leu Asp Asp Leu 85 90 95 Asp Ala Cys Leu Gly Gln Val Thr GlyGlu Glu Asp Ser Ala Leu Gly 100 105 110 Arg Thr Gly Pro Thr Leu Ala MetLys Arg Tyr Phe Gln Gly Ile His 115 120 125 Val Tyr Leu Lys Glu Lys GlyTyr Ser Asp Cys Ala Trp Glu Ile Val 130 135 140 Arg Leu Glu Ile Met ArgSer Leu Ser Ser Ser Thr Ser Leu His Lys 145 150 155 160 Arg Leu Arg MetMet Asp Gly Asp Leu Ser Ser Pro 165 170 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 amino acids (B) TYPE:amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment1-37 of SEQ ID NO:33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Cys AspLeu Ser Gln Asn His Val Leu Val Gly Ser Gln Asn Leu Arg 1 5 10 15 LeuLeu Gly Gln Met Arg Arg Leu Ser Leu Arg Phe Cys Leu Gln Asp 20 25 30 ArgLys Asp Phe Ala 35 (2) INFORMATION FOR SEQ ID NO: 36: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: Amino acid sequence of fragment 34-64 of SEQ IDNO:33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Lys Asp Phe Ala Phe ProGln Glu Met Val Glu Gly Gly Gln Leu Gln 1 5 10 15 Glu Ala Gln Ala IleSer Val Leu His Glu Met Leu Gln Gln Ser 20 25 30 (2) INFORMATION FOR SEQID NO: 37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi)ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Amino acid sequence of fragment62-92 of SEQ ID NO:33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: Gln GlnSer Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp 1 5 10 15 AspThr Thr Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gln Gln 20 25 30 (2)INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:33 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Amino acidsequence of fragment 90-122 of SEQ ID NO:33 (xi) SEQUENCE DESCRIPTION:SEQ ID NO: 38: His Gln Gln Leu Asp Asp Leu Asp Ala Cys Leu Gly Gln ValThr Gly 1 5 10 15 Glu Glu Asp Ser Ala Leu Gly Arg Thr Gly Pro Thr LeuAla Met Lys 20 25 30 Arg (2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 32 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: Amino acid sequence of fragment 119-150 of SEQ IDNO:33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: Ala Met Lys Arg Tyr PheGln Gly Ile His Val Tyr Leu Lys Glu Lys 1 5 10 15 Gly Tyr Ser Asp CysAla Trp Glu Ile Val Arg Leu Glu Ile Met Arg 20 25 30 (2) INFORMATION FORSEQ ID NO: 40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: Amino acid sequence offragment 139-172 of SEQ ID NO:33 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: Cys Ala Trp Glu Ile Val Arg Leu Glu Ile Met Arg Ser Leu Ser Ser 1 510 15 Ser Thr Ser Leu His Lys Arg Leu Arg Met Met Asp Gly Asp Leu Ser 2025 30 Ser Pro (2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (D)TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C)INDIVIDUAL ISOLATE: Amino acid sequence of fragment 1-23 of SEQ ID NO:32(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: Met Ala Phe Val Leu Ser LeuLeu Met Ala Leu Val Leu Val Ser Tyr 1 5 10 15 Gly Pro Gly Gly Ser LeuArg 20 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (C) INDIVIDUALISOLATE: Amino acid sequence of fragment 1-23 of SEQ ID NO:11 (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 42: Met Ala Phe Val Leu Ser Leu Leu MetAla Leu Val Leu Val Ser Tyr 1 5 10 15 Gly Pro Gly Gly Ser Leu Gly 20 (2)INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:519 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE:HuIFNtau1 genomic-derived DNA coding sequence, without leader seq. (ix)FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..519 (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 43: TGT GAC CTG TCT CAG AAC CAC GTG CTG GTT GGCAGG AAG AAC CTC AGG 48 Cys Asp Leu Ser Gln Asn His Val Leu Val Gly ArgLys Asn Leu Arg 1 5 10 15 CTC CTG GAC GAA ATG AGG AGA CTC TCC CCT CGCTTT TGT CTG CAG GAC 96 Leu Leu Asp Glu Met Arg Arg Leu Ser Pro Arg PheCys Leu Gln Asp 20 25 30 AGA AAA GAC TTC GCT TTA CCC CAG GAA ATG GTG GAGGGC GGC CAG CTC 144 Arg Lys Asp Phe Ala Leu Pro Gln Glu Met Val Glu GlyGly Gln Leu 35 40 45 CAG GAG GCC CAG GCC ATC TCT GTG CTC CAT GAG ATG CTCCAG CAG AGC 192 Gln Glu Ala Gln Ala Ile Ser Val Leu His Glu Met Leu GlnGln Ser 50 55 60 TTC AAC CTC TTC CAC ACA GAG CAC TCC TCT GCT GCC TGG GACACC ACC 240 Phe Asn Leu Phe His Thr Glu His Ser Ser Ala Ala Trp Asp ThrThr 65 70 75 80 CTC CTG GAG CAG CTC CGC ACT GGA CTC CAT CAG CAG CTG GACAAC CTG 288 Leu Leu Glu Gln Leu Arg Thr Gly Leu His Gln Gln Leu Asp AsnLeu 85 90 95 GAT GCC TGC CTG GGG CAG GTG ATG GGA GAG GAA GAC TCT GCC CTGGGA 336 Asp Ala Cys Leu Gly Gln Val Met Gly Glu Glu Asp Ser Ala Leu Gly100 105 110 AGG ACG GGC CCC ACC CTG GCT CTG AAG AGG TAC TTC CAG GGC ATCCAT 384 Arg Thr Gly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gln Gly Ile His115 120 125 GTC TAC CTG AAA GAG AAG GGA TAC AGC GAC TGC GCC TGG GAA ACCGTC 432 Val Tyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val130 135 140 AGA CTG GAA ATC ATG AGA TCC TTC TCT TCA TTA ATC AGC TTG CAAGAA 480 Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Leu Ile Ser Leu Gln Glu145 150 155 160 AGG TTA AGA ATG ATG GAT GGA GAC CTG AGC TCA CCT TGA 519Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170 (2) INFORMATIONFOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 172 aminoacids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Cys Asp Leu Ser GlnAsn His Val Leu Val Gly Arg Lys Asn Leu Arg 1 5 10 15 Leu Leu Asp GluMet Arg Arg Leu Ser Pro Arg Phe Cys Leu Gln Asp 20 25 30 Arg Lys Asp PheAla Leu Pro Gln Glu Met Val Glu Gly Gly Gln Leu 35 40 45 Gln Glu Ala GlnAla Ile Ser Val Leu His Glu Met Leu Gln Gln Ser 50 55 60 Phe Asn Leu PheHis Thr Glu His Ser Ser Ala Ala Trp Asp Thr Thr 65 70 75 80 Leu Leu GluGln Leu Arg Thr Gly Leu His Gln Gln Leu Asp Asn Leu 85 90 95 Asp Ala CysLeu Gly Gln Val Met Gly Glu Glu Asp Ser Ala Leu Gly 100 105 110 Arg ThrGly Pro Thr Leu Ala Leu Lys Arg Tyr Phe Gln Gly Ile His 115 120 125 ValTyr Leu Lys Glu Lys Gly Tyr Ser Asp Cys Ala Trp Glu Thr Val 130 135 140Arg Leu Glu Ile Met Arg Ser Phe Ser Ser Leu Ile Ser Leu Gln Glu 145 150155 160 Arg Leu Arg Met Met Asp Gly Asp Leu Ser Ser Pro 165 170

It is claimed:
 1. An isolated nucleic acid molecule that encodes anovine interferon-τ.
 2. A nucleic acid of claim 1, where said nucleicacid molecule has the sequence presented as SEQ ID NO:1.
 3. A nucleicacid molecule of claim 1, wherein said nucleic acid molecule encodes apolypeptide having a sequence presented as SEQ ID NO:2.
 4. A nucleicacid of claim 3, where said polypeptide includes a leader sequence. 5.An expression vector comprising (a) a nucleic acid containing an openreading frame that encodes the ovine interferon-τ; and (b) regulatorysequences effective to express said open reading frame in a host cell.6. A method of recombinantly producing ovine interferon-τ, comprisingintroducing into suitable host cells, a recombinant expression systemcontaining an open reading frame (ORF) having a polynucleotide sequencewhich encodes an ovine interferon-τ polypeptide, where the vector isdesigned to express the ORF in said host, and culturing said host underconditions resulting in the expression of the ORF sequence.
 7. Arecombinantly produced ovine interferon-τ protein.
 8. The recombinantlyproduced protein of claim 7, where said protein has the sequencepresented as SEQ ID NO:2.
 9. A method of inhibiting tumor cell growth,comprising contacting the cells with ovine interferon-τ at aconcentration effective to inhibit growth of the tumor cells.
 10. Amethod of inhibiting viral replication, comprising contacting cellsinfected with a virus with ovine interferon-τ at a concentrationeffective to inhibit viral replication within said cells.
 11. The methodof claim 10, where said virus is an RNA virus.
 12. The method of claim11, where said virus is selected from the group consisting of felineleukemia virus, ovine lentivirus, equine infectious anemia virus, bovineimmunodeficiency virus, visna-maedi virus, and caprine arthritisencephalitis.
 13. The method of claim 10, where said virus is a DNAvirus.
 14. The method of claim 10, where said interferon-τ has theprotein sequence presented as SEQ ID NO:2.
 15. An isolated nucleic acidmolecule that encodes a human interferon-τ.
 16. A nucleic acid of claim15, where said nucleic acid molecule includes the sequence presented asSEQ ID NO:43.
 17. A nucleic acid molecule of claim 15, wherein saidnucleic acid molecule encodes a polypeptide having a sequence presentedas SEQ ID NO:44.
 18. A nucleic acid molecule of claim 17, where saidpolypeptide further includes a leader sequence.
 19. A nucleic acid ofclaim 15, where said nucleic acid molecule includes the sequencepresented as SEQ ID NO:29.
 20. A nucleic acid molecule of claim 15,wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:30.
 21. A nucleic acid molecule of claim20, where said polypeptide further includes a leader sequence.
 22. Anucleic acid of claim 15, where said nucleic acid molecule includes thesequence presented as SEQ ID NO:33.
 23. A nucleic acid molecule of claim15, wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:34.
 24. A nucleic acid molecule of claim23, where said polypeptide further includes a leader sequence.
 25. Anucleic acid of claim 15, where said nucleic acid molecule includes thesequence presented as SEQ ID NO:25.
 26. A nucleic acid molecule of claim15, wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:26.
 27. A nucleic acid molecule of claim26, where said polypeptide further includes a leader sequence.
 28. Anucleic acid of claim 15, where said nucleic acid molecule includes thesequence presented as SEQ ID NO:27.
 29. A nucleic acid molecule of claim15, wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:28.
 30. A nucleic acid molecule of claim29, where said polypeptide further includes a leader sequence.
 31. Anucleic acid of claim 15, where said nucleic acid molecule includes thesequence presented as SEQ ID NO:21.
 32. A nucleic acid molecule of claim15, wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:22.
 33. A nucleic acid molecule of claim32, where said polypeptide further includes a leader sequence.
 34. Anucleic acid of claim 15, where said nucleic acid molecule includes thesequence presented as SEQ ID NO:23.
 35. A nucleic acid molecule of claim15, wherein said nucleic acid molecule encodes a polypeptide having asequence presented as SEQ ID NO:24.
 36. A nucleic acid molecule of claim35, where said polypeptide further includes a leader sequence.
 37. Anexpression vector comprising (a) a nucleic acid containing an openreading frame that encodes a human interferon-τ; and (b) regulatorysequences effective to express said open reading frame in a host cell.38. An expression vector of claim 37, where said human interferon-τcontains a polypeptide having a sequence presented as SEQ ID NO:44. 39.An expression vector of claim 37, where said human interferon-τ containsa polypeptide having a sequence presented as SEQ ID NO:30.
 40. Anexpression vector of claim 37, where said human interferon-τ contains apolypeptide having a sequence presented as SEQ ID NO:34.
 41. Anexpression vector of claim 37, where said human interferon-τ contains apolypeptide having a sequence presented as SEQ ID NO:26.
 42. Anexpression vector of claim 37, where said human interferon-τ contains apolypeptide having a sequence presented as SEQ ID NO:28.
 43. Anexpression vector of claim 37, where said human interferon-τ contains apolypeptide having a sequence presented as SEQ ID NO:22.
 44. Anexpression vector of claim 37, where said human interferon-τ contains apolypeptide having a sequence presented as SEQ ID NO:24.
 45. A method ofrecombinantly producing human interferon-τ, comprising introducing intosuitable host cells, a recombinant expression system containing an openreading frame (ORF) having a polynucleotide sequence which encodes ahuman interferon-τ polypeptide, where the vector is designed to expressthe ORF in said host, and culturing said host under conditions resultingin the expression of the ORF sequence.
 46. An isolated humaninterferon-τ protein.
 47. A protein of claim 46, where said protein isrecombinantly produced.
 48. A protein of claim 46, where said proteincontains the sequence presented as SEQ ID NO:44.
 49. A protein of claim46, where said protein contains the sequence presented as SEQ ID NO:30.50. A protein of claim 46, where said protein contains the sequencepresented as SEQ ID NO:34.
 51. A protein of claim 46, where said proteincontains the sequence presented as SEQ ID NO:26.
 52. A protein of claim46, where said protein contains the sequence presented as SEQ ID NO:28.53. A protein of claim 46, where said protein contains the sequencepresented as SEQ ID NO:22.
 54. A protein of claim 46, where said proteincontains the sequence presented as SEQ ID NO:24.
 55. A method ofinhibiting tumor cell growth, comprising contacting the cells with humaninterferon-τ at a concentration effective to inhibit growth of the tumorcells.
 56. A method of claim 55, wherein said cells are human carcinomacells, human leukemia cells, human T-lymphoma cells, and human melanomacells.
 57. A method of claim 56, wherein said cells aresteroid-sensitive tumor cells.
 58. A method of claim 57, wherein saidcells are mammary tumor cells.
 59. A method of claim 55, where saidprotein contains the sequence presented as SEQ ID NO:44.
 60. A method ofclaim 55, where said protein contains the sequence presented as SEQ IDNO:30.
 61. A method of claim 55, where said protein contains thesequence presented as SEQ ID NO:34.
 62. A method of claim 55, where saidprotein contains the sequence presented as SEQ ID NO:26.
 63. A method ofclaim 55, where said protein contains the sequence presented as SEQ IDNO:28.
 64. A method of claim 55, where said protein contains thesequence presented as SEQ ID NO:22.
 65. A method of claim 55, where saidprotein contains the sequence presented as SEQ ID NO:24.
 66. A method ofinhibiting viral replication, comprising contacting cells infected witha virus with human interferon-τ at a concentration effective to inhibitviral replication within said cells.
 67. A method of claim 66, wheresaid virus is an RNA virus.
 68. A method of claim 67, where said virusis human immunodeficiency virus, or hepatitis c virus.
 69. A method ofclaim 66, where said virus is a DNA virus.
 70. A method of claim 69,where said virus is hepatitis B virus.
 71. A method of claim 66, wheresaid protein contains the sequence presented as SEQ ID NO:44.
 72. Amethod of claim 66, where said protein contains the sequence presentedas SEQ ID NO:30.
 73. A method of claim 66, where said protein containsthe sequence presented as SEQ ID NO:34.
 74. A method of claim 66, wheresaid protein contains the sequence presented as SEQ ID NO:26.
 75. Amethod of claim 66, where said protein contains the sequence presentedas SEQ ID NO:28.
 76. A method of claim 66, where said protein containsthe sequence presented as SEQ ID NO:22.
 77. A method of claim 66, wheresaid protein contains the sequence presented as SEQ ID NO:24.
 78. Amethod of enhancing fertility in a female mammal, comprisingadministering to said mammal an effective mammalian fertility enhancingamount of human interferon-τ in a pharmaceutically acceptable carrier.79. A method of claim 78, where said protein contains the sequencepresented as SEQ ID NO:44.
 80. A method of claim 78, where said proteincontains the sequence presented as SEQ ID NO:30.
 81. A method of claim78, where said protein contains the sequence presented as SEQ ID NO:34.82. A method of claim 78, where said protein contains the sequencepresented as SEQ ID NO:26.
 83. A method of claim 78, where said proteincontains the sequence presented as SEQ ID NO:28.
 84. A method of claim78, where said protein contains the sequence presented as SEQ ID NO:22.85. A method of claim 78, where said protein contains the sequencepresented as SEQ ID NO:24.
 86. A fused polypeptide, comprising: (a) aninterferon-τ polypeptide, where said polypeptide is (i) derived from aninterferon-τ amino acid coding sequence, and (ii) between 15 and 172amino acids long; and (b) a second soluble polypeptide.
 87. The fusedpolypeptide of claim 86, wherein said interferon-τ polypeptide isselected from the group consisting of SEQ ID NO:5 and SEQ ID NO:15. 88.The fused polypeptide of claim 86, wherein said second solublepolypeptide is interferon-α.
 89. The fused polypeptide of claim 86,wherein said second soluble polypeptide is interferon-β.
 90. Aninterferon-τ/type I interferon fusion protein comprising: a type Iinterferon protein wherein the N-terminal cytotoxic region of said typeI interferon is replaced by an N-terminal region of interferon-τ withina sequence spanning residues 1 to 37 of interferon-τ, and the fusionprotein has reduced cytotoxicity relative to the cytotoxicity of thetype I interferon.
 91. The fusion protein of claim 90, wherein said typeI interferon is interferon-α.
 92. The fusion protein of claim 90,wherein said type I interferon is interferon-β.
 93. The fusion proteinof claim 90, wherein said interferon-τ is ovine, bovine, or humaninterferon-τ.
 94. The fusion protein of claim 90, wherein the sequencefrom residues 1 to 37 is SEQ ID NO:5 and the sequence from residues 38to the C-terminal residue is the corresponding sequence of said type Iinterferon.
 95. The fusion protein of claim 90, wherein the sequencefrom residues 1 to 37 is SEQ ID NO:15 and the sequence from residues 38to the C-terminal residue is the corresponding sequence of said type Iinterferon.